Microfluidic platform for multiplexed detection in single cells and methods thereof

ABSTRACT

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/918,402, filed Dec. 19, 2013, which is hereby incorporated byreference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under contract no.DE-AC04-94AL85000 awarded by the U.S. Department of Energy to SandiaCorporation. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a microfluidic device and platformconfigured to conduct multiplexed analysis within the device (i.e.,on-chip). Also provided are methods of performing multiplexed analysesto detect one or more target nucleic acids, proteins, andpost-translational modifications.

BACKGROUND OF THE INVENTION

Cellular signaling networks include complex reactions between numerousbiomolecules, such as RNA, DNA, and proteins. In addition, individualcells behave differently, further necessitating tools that can detectdifferent types of biomolecules on a single-cell level. Studies ofsignaling networks can elucidate and decipher the molecular mechanismsof cellular and disease processes. Such a study requires an integrated,multiplexed platform that enables a systems-level investigation of thecomplex interactions between these signaling biomolecules. To date, notechnology exists that can integrate the detection and analysis of allthe signaling molecules, particularly at the single-cell level. Thus,additional tools and systems are desired to perform such single-cellstudies.

SUMMARY OF THE INVENTION

The present invention relates to an automated microfluidic platform forperforming multiplexed molecular assays for simultaneous detection ofmicroRNA, mRNA, DNA, and/or proteins (e.g., including post-translationmodified proteins) in single cells. In one example, an exemplarymicrofluidic platform herein is configured to conduct a portfolio ofcustomized molecular assays that can detect nucleic acids, proteins, andpost-translational modifications in single intact cells with >95%reduction in reagent requirement in under 8 hours (h.).

Accordingly, in a first aspect, the invention features a method forperforming multiplexed analysis in a microfluidic device, the methodincluding: (i) loading a test sample portion into a main port of thedevice, where the device includes a plurality of assay chambers and themain port is configured to deliver the test sample portion to each assaychamber; (ii) capturing the test sample portion within each assaychamber, thereby providing a captured sample portion; (iii) incubatingthe captured sample portion with a first protein label in each assaychamber, where the first protein label is configured to detect a firsttarget protein (e.g., a cell surface target), thereby providing alabeled sample portion; (iv) treating the labeled sample portion with afixative reagent in each assay chamber, thereby providing a fixed sampleportion; (v) incubating the fixed sample portion with a first nucleicacid label, where the first nucleic acid label is configured to detect afirst target nucleic acid, thereby providing a multiplexed-labeledsample portion; (vi) detaching the multiplexed-labeled sample portion,thereby providing a detached sample portion; and (vii) performing anon-chip flow cytometry assay of the detached sample portion, therebyperforming multiplexed analysis for the target protein and/or the targetnucleic acid.

In some embodiments, the method further includes (e.g., after step (v))amplifying a signal of the first and/or second nucleic acid label. Infurther embodiments, the amplifying step includes performing a rollingcircle amplification, an isothermal amplification, or any other nucleicacid amplification methodology by providing one or more affinity agentsconfigured to bind to the first and/or second nucleic acid label, one ormore proximity ligation probes configured to bind at least one affinityagent, one or more connector probes configured to bind at least oneproximity ligation probe and to form a circular template, and/or one ormore enzymes configured to generate a concatemer based on the circulartemplate.

In a second aspect, the invention features a method for performingmultiplexed analysis in a microfluidic device, the method including: (i)loading a test sample portion into a main port of the device, where thedevice includes a plurality of assay chambers and the main port isconfigured to deliver the test sample portion to each assay chamber;(ii) capturing the test sample portion within each assay chamber,thereby providing a captured sample portion; (iii) treating the capturedsample portion with a first fixative reagent in each assay chamber,thereby providing a fixed sample portion; (iv) incubating the fixedsample portion with a first protein label in each assay chamber, wherethe first protein label is configured to detect a first target protein,thereby providing a labeled sample portion; (v) incubating the labeledsample portion with a second protein label in each assay chamber, wherethe second protein label is configured to detect a second target proteinand the second target protein is different than the first targetprotein, thereby providing a multi-labeled sample portion; (vi)incubating the multi-labeled sample portion with a first nucleic acidlabel, where the first nucleic acid label is configured to detect afirst target nucleic acid, thereby providing a multiplexed-labeledsample portion; (vii) amplifying a signal of the first nucleic acidlabel, thereby providing an amplified sample portion; (viii) detachingthe amplified sample portion, thereby providing a detached sampleportion; and (ix) performing an on-chip flow cytometry assay of thedetached sample portion, thereby performing multiplexed analysis for thetarget proteins and the target nucleic acid.

In a third aspect, the invention feature method for performingmultiplexed analysis in a microfluidic device, the method including: (i)loading a test sample portion into a main port of the device, where thedevice includes a plurality of assay chambers and the main port isconfigured to deliver the test sample portion to each assay chamber;(ii) capturing the test sample portion within each assay chamber,thereby providing a captured sample portion; (iii) incubating thecaptured sample portion with a first protein label for a cell surfacetarget in each assay chamber, where the first protein label isconfigured to detect a first target protein, thereby providing a labeledsample portion; (iv) treating the labeled sample portion with a fixativereagent in each assay chamber, thereby providing a fixed sample portion;(v) treating the fixed sample with a permeabilization reagent to provideaccess to an intracellular target, thereby providing a fixed,permeabilized sample; (vi) treating the fixed, permeabilized sample withone or more target protein labels for detecting one or moreintracellular target proteins; (vii) treating the fixed, permeabilizedsample (e.g., after step (v) and/or (vi) with one or more fixativereagents to prepare the sample for nucleic acid hybridization (e.g.,where the fixative reagent for step (vii) can be the same or differentas the fixative reagent in step (iv)); (viii) incubating the fixed,permeabilized sample (e.g., after step (v), (vi), and/or (vii)) with oneor more nucleic acid labels, where the nucleic acid labels areconfigured to detect a multiple target nucleic acids (e.g., includingsmall RNAs, long RNAs, and DNAs), thereby providing amultiplexed-labeled sample portion; (ix) performing signal amplificationusing rolling circle polymerase (e.g., to detect rare nucleic acidshybridized to one or more nucleic acid labels or probes); (x) detachingthe multiplexed-labeled sample portion (e.g., after step (ix)), therebyproviding a detached sample portion; and (xi) performing an on-chip flowcytometry assay of the detached sample portion, thereby performingmultiplexed analysis for the target protein and/or the target nucleicacids.

In any method herein, the method further includes (e.g., after step (i)or (ii)) culturing the test sample portion and/or stimulating the testsample portion with a stimulant.

In any method herein, the method further includes (e.g., after step(iii)) incubating the labeled sample portion with a secondary labelincluding a second affinity agent and a detectable marker, where thefirst and second affinity agents are configured to bind together.

In any method herein, the method further includes (e.g., after step (v))incubating the multiplexed-labeled sample portion with a secondary labelincluding a second affinity agent, where the first and second affinityagents are configured to bind together.

In any method herein, the method further includes incubating themultiplexed-labeled sample portion with one or more tertiary labels,where each tertiary label is, independently, configured to bind to thesecond label and independently includes a proximity ligation probe;and/or incubating with one or more quaternary labels, where eachquaternary label is, independently, configured to bind to a portion ofthe proximity ligation probe.

In any method herein, the method further includes (e.g., during or afteran incubating step) incubating the fixed sample portion with a secondnucleic acid label to detect a second target nucleic acid.

In any method herein, the method further includes treating the capturedsample portion, the labeled sample portion, fixed sample portion, and/ormultiplexed-labeled sample portion with a fixative reagent, where eachfixative reagent can be the same or different.

In any method herein, the method further includes incubating the labeledsample portion with one or more other protein labels (e.g., a secondprotein label) in each assay chamber, where each protein label isconfigured, independently, to detect a target protein, thereby providingthe labeled sample portion.

In any method herein, the method further includes incubating thecaptured sample portion, the labeled sample portion, the fixed sampleportion, and/or the multiplexed-labeled sample portion with apost-translation modification label (e.g., any herein).

In any method herein, the method further includes treating themulti-labeled sample portion with a second fixative reagent (e.g., wherethe first and second fixative reagents can be the same or different),thereby providing the multi-labeled sample portion.

In any method herein, the method further includes permeabilizing thelabeled sample portion with a permeabilization reagent.

In any method herein, the performing an on-chip flow cytometry assaystep includes transporting the detached sample portion to a flowcytometry channel of the device, hydrodynamically focusing the detachedsample portion by employing one or more sheath fluids, and detecting oneor more protein labels and/or nucleic acid labels by applying anexcitation source to the hydrodynamically focused sample portion.

In a fourth aspect, the invention features a microfluidic platform formultiplexed analysis, where the platform includes a microfluidic device,a manifold coupled to the device, a pumping system coupled to themanifold, a controller coupled to the pumping system and configured tocontrol the pumping system, a stage coupled to the device, an excitationsource configured to provide an excitation energy to the flow cytometrychannel, and a detector configured to receive an emission spectrum froman excited label within the device.

In some embodiments, the microfluidic device includes a plurality ofassay chambers, where each assay chamber is configured to conduct amultiplexed series of assays and each assay chamber is individuallyaddressable; a main port in fluidic communication with each assaychamber, where the main port is configured to deliver a test sampleportion to each assay chamber; a plurality of ports, where each assaychamber is in fluidic communication with at least one port; a flowcytometry channel in fluidic communication with each assay chamber,where the flow cytometry channel is configured to hydrodynamically focusthe test sample portion; and one or more sheath ports in fluidiccommunication with the flow cytometry channel, where the sheath port isconfigured to deliver a sheath fluid to the flow cytometry channel.

In some embodiments, the manifold includes a first reservoir configuredto contain the test sample portion, where the first reservoir is influidic communication with the main port; a second reservoir configuredto contain a first protein label, where the second reservoir is influidic communication with at least one port or the main port and wherethe first protein label is configured to detect a first target protein;a third reservoir configured to contain a first fixative reagent, wherethe third reservoir is in fluidic communication at least one port or themain port; a fourth reservoir configured to contain a first nucleic acidlabel, where the fourth reservoir is in fluidic communication with atleast one port or the main port and where the first nucleic acid labelis configured to detect a first target nucleic acid; and a fifthreservoir configured to contain one or more sheath fluids, where thefifth reservoir is in fluidic communication with the sheath port. Infurther embodiments, the manifold further includes a sixth reservoirconfigured to contain one or more amplification reagents, where thesixth reservoir is in fluidic communication with at least one port orthe main port and where the one or more amplification reagents areconfigured to amplify a signal of the first nucleic acid label.

In some embodiments, the manifold further includes one or more valves(e.g., a valve disposed between the first reservoir and the main port;and/or a valve disposed between each reservoir and the port in fluidiccommunication with each reservoir). In other embodiments, the controlleris further configured to operate the valve.

In some embodiments, the pumping system is coupled to the first, second,third, fourth, and fifth reservoirs. In other embodiments, the pumpingsystem further includes a pressure source and a pressure controllerconfigured to be controlled by the controller.

In some embodiments, the controller is configured to execute any methodherein. In one embodiment, the controller is configured to execute thefollowing: fluidically deliver the test sample portion from the firstreservoir to the main port and then to each assay chamber; fluidicallydeliver the first protein label from the second reservoir to at leastone assay chamber, thereby providing a labeled sample portion;fluidically deliver the first fixative reagent from the third reservoirto at least one assay chamber containing the labeled sample portion,thereby providing a fixed sample portion; fluidically deliver the firstnucleic acid label from the fourth reservoir to at least one assaychamber containing the fixed sample portion, thereby providing amulti-labeled sample portion; fluidically deliver the multi-labeledsample portion(s) from at least one assay chamber to the flow cytometrychannel; and fluidically deliver one or more sheath fluids from thefifth reservoir to the sheath port. In further embodiments, thecontroller is further configured to fluidically deliver the one or moreamplification reagents to the multi-labeled sample portion prior to themulti-labeled sample portion being delivered to the flow cytometrychannel.

In some embodiments, the stage is configured to control a temperaturewithin the plurality of assay chambers.

In some embodiments, the excitation source is configured to excite oneor more labels (e.g., the first protein label, the first nucleic acidlabel, or a secondary label configured to directly or indirectly bind tothe first protein label or the first nucleic label), thereby providingan excited label for the multi-labeled sample portion.

In any embodiment herein, the first protein label and/or the firstnucleic acid label further includes a detectable marker. In someembodiments, the first protein label further includes a first affinityagent. In other embodiments, the first nucleic acid label furtherincludes a first affinity agent.

In any embodiment herein, each of the first and nucleic acid labelsincludes, independently, a nucleic acid-binding region configured todetect the target nucleic acid and one or more affinity agents.

In any embodiment herein, the first target protein is a cell surfaceprotein, and the second target protein is an intercellular protein.

In any embodiment herein, the fixative reagent includes formaldehyde,paraformaldehyde, glutaraldehyde,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), and/orN-hydroxysulfosuccinimide (sulfo-NHS), as well as any other describedherein.

Definitions

As used herein, the term “about” means +/−10% of any recited value. Asused herein, this term modifies any recited value, range of values, orendpoints of one or more ranges.

By “binding” is meant having a covalent or a non-covalent bond, such asone or more of electrostatic, ionic, hydrogen, van der Waals, π-effect,π-bonding, or hydrophobic bonds.

By “coupled” is meant indirect or direct contact between two or morecomponents or structures, thereby allowing at least two components tointeract. In particular embodiment, two or more components are coupledin a reversible manner.

By “chamber” is meant a two-dimensional or three-dimensional region ofthe microfluidic device configured to confine a fluid (e.g., a gas, aliquid, a colloid, a solution, etc.). This region may be enclosed withina substrate and include one or more ports or channels in fluidiccommunication with that region. Alternatively, the region can have anopen format and may not be enclosed. The chamber can have any usefulconfiguration (e.g., a channel, a well, a tube, a pipe, etc.) anddimension (e.g., a microchamber or nanochamber, including microchannels,nanochannels, microwell, and nanowells) having one or more optionalports.

By “fluidic communication,” as used herein, refers to any duct, channel,tube, pipe, chamber, or pathway through which a substance, such as aliquid, gas, or solid may pass substantially unrestricted when thepathway is open. When the pathway is closed, the substance issubstantially restricted from passing through. Typically, limiteddiffusion of a substance through the material of a plate, base, and/or asubstrate, which may or may not occur depending on the compositions ofthe substance and materials, does not constitute fluidic communication.

By “microfluidic” or “micro” is meant having at least one dimension thatis less than 1 mm. For instance, a microfluidic structure (e.g., anystructure described herein) can have a length, width, height,cross-sectional dimension, circumference, radius (e.g., external orinternal radius), or diameter that is less than 1 mm.

By “protein” is meant a polymer of two or more amino acids (natural,unnatural, or modified amino acids) linked together. As used herein,“protein” is used interchangeably with “polypeptide” and “peptide.”

By “nucleic acid” is meant a polymer of two or more nucleotides. As usedherein, a “nucleic acid” is used interchangeably with “polynucleotide.”Exemplary nucleic acids include, but are not limited to, ribonucleicacids (RNAs) (e.g., a messenger RNA (mRNA), a micro RNA (miRNA), a longnoncoding RNA (lncRNA or lincRNA), a small nucleolar RNA (sno-RNA), asmall interfering RNA (siRNA), or a Piwi-interacting RNA (piRNA), orportions thereof); deoxyribonucleic acids (DNAs); threose nucleic acids(TNAs); glycol nucleic acids (GNAs); peptide nucleic acids (PNAs);locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization); or hybrids thereof.In particular embodiments, the nucleic acid or nucleic acid sequenceselectively hybridize to another nucleic acid sequence. Such specifichybridization can include, e.g., binding, duplexing, or hybridizing of anucleic acid preferentially to a particular nucleic acid sequence understringent conditions when that sequence is present in a complex mixture(e.g., total cellular) of nucleic acids (e.g., DNA and/or RNA mixtures).In some embodiments, stringent conditions refer to conditions underwhich a probe will hybridize preferentially to its target sequence, andto a lesser extent to, or not at all to, other sequences. Exemplary,non-limiting stringent conditions include highly stringent hybridizationand wash conditions that are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH, where T_(m) is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa perfectly matched probe, such as wash conditions of 0.15 M NaCl at 72°C. for about 15 min.; 0.2×SSC at 65° C. for 15 min.; 1×SSC wash at 45°C. for 15 min.; and/or 4-6×SSC at 40° C. for 15 min., where these washescan be combined in any useful sequence.

As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,”and “below” are used to provide a relative relationship betweenstructures. The use of these terms does not indicate or require that aparticular structure must be located at a particular location in theapparatus.

Other features and advantages of the invention will be apparent from thefollowing description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C provides exemplary schematics of a microfluidic device 100and microfluidic platforms 1000, 1100.

FIG. 2A-2D provides schematics of (A) an exemplary microfluidic device;(B) a device coupled to a stage and in fluidic communication with amanifold; (C) a platform including various controllers, pumps, andvalves; and (D) the optical detector and excitation source pathway forthe platform.

FIG. 3A-3C provides exemplary devices and platforms. Shown are (A) anexemplary schematic of a ten-chamber microfluidic device 300 (left) anda microphotograph of the flow cytometry region (right), in which cellsprepared in each of the assay chambers can be detached and driven to thecenter of the chip for hydrodynamic focus and flow cytometry; (B) anexemplary schematic of a platform 3000; and (C) an exemplary schematicof an optical system for detection 3100.

FIG. 4A-4B provides schematics of (A) an exemplary nucleic acid label410 configured to detect the target nucleic acid 401 and (B) anexemplary reaction between a target miRNA, a cross-linker (e.g., EDC),and a nearby amino group (e.g., provided by a nearby protein).

FIG. 5A-5D provides Flow-FISH detection of a target nucleic acid(miR155). Shown are (A) a schematic of the microfluidic device and assayconditions; (B) a graph showing the frequency of miR155-labeled cells,which were detected by fluorescence and flow cytometry, afterstimulation with PMA and ionomycin for 0, 8, 16, 20, or 24 h.; (C) agraph showing median miR155 fluorescence after stimulation for 0, 8, 16,20, or 24 h., where * indicates p<0.01 and ** indicates p>0.01; and (D)a graph showing fold change in RT-qPCR amplicons after stimulation for0, 8, 16, 20, or 24 h., where ** indicates p>0.01 and * indicatesp<0.01.

FIG. 6A-6C provides on-chip multiplexed detection of a target nucleicacid (miR155) and a target protein (CD69). Shown are (A) fluorescencemicrophotographs of Jurkat cells showing RCA-amplified miR155 signals inthe cytosol (shown as dots) and CD69 protein stained with quantum dots(peripheral edges indicated by white arrowheads); (B) a graph showingmedian miR155 fluorescence (diamonds) and median CD69 fluorescence(circles) collected via on-chip flow cytometry; and (C) a scatter plotshowing heterogeneity of miR155 and CD69 expression in individual Jurkatcells after 24 hours stimulation with PMA and ionomycin. Each cell isindicated by a data point (diamond).

FIG. 7 is a schematic of a cell 700 showing various cellular processesand exemplary targets suitable for multiplexed detection using themethods, devices, and platforms described herein. In one exemplaryprocess, activation of a cell surface receptor 701 leads to aphosphorylation signaling cascade 702, which then induces thetranscription of messenger RNAs (mRNAs) and microRNAs (miRNAs) 703. ThemRNA and miRNA are exported into the cytosol, and mRNA is translatedinto proteins 705. The miRNA can inhibit protein translation by bindingmRNA and inducing their degradation 704.

FIG. 8A-8F provides a portfolio of microfluidic assays developed for theplatform herein. Shown are (A) microRNA (miRNA) analysis; (B) messengerRNA (mRNA) analysis; (C) cell surface protein analysis; (D) proteinphosphorylation profiles; (E) cytosolic protein expression profiles; and(F) dynamic glycosylation profiles that were performed on-chip. Providedare data of (A) miRNA 155 in Jurkat cells after 24 h. stimulation withPMA and ionomycin; (B) β-actin mRNA in Jurkat cells compared with ascrambled control probe; (C) RAW 264.7 cell surface receptor activationas demonstrated by TLR4-MD2 receptor activation by lipopolysaccharide(LPS) at 30 s. and 30 min.; (D) transient P38 protein phosphorylation inRAW 264.7 upon 30 min. stimulation with LPS; (E) cytosolic tumornecrosis factor-α (TNF-α) cytokine production in LPS-stimulated RAW264.7 cells; and (F) dynamic glycosylation of nucleoporin 62 (Nup62)after stimulation.

FIG. 9 shows a diagram of an exemplary method 900 for performingmultiplexed analysis, as described herein, employing a first proteinlabel and a nucleic acid label.

FIG. 10 shows a diagram of an exemplary method 1000 for performingmultiplexed analysis, as described herein, employing a first proteinlabel, a second protein label, and a nucleic acid label.

FIG. 11 shows a diagram of an exemplary method 1100 for performingmultiplexed analysis, as described herein, employing various fixativereagents, permeabilization reagents, and labels.

FIG. 12 shows a diagram of an exemplary method 1200 for performingmultiplexed analysis of miRNA, mRNA, and proteins under 8 hours, asdescribed herein, while employing minimal sample amounts (e.g., lessthan 5000 cells, such as of from about 1000 to 3000 cells) and minimalreagent volumes (e.g., less than about 300 nL, such as about 270 nL).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to microfluidic devices and platformsconfigured to perform multiplexed analysis with a single-cellresolution. In particular, distinct levels of various nucleic acids(e.g., mRNA, DNA, miRNA, etc.) and proteins (e.g., cell surfaceproteins, intracellular proteins, and/or post-translation modifiedproteins) can be detected in each single, intact cell. This capabilityis facilitated by a microfluidic device configured to capture and assayindividual cells, and then to detect target levels using an on-chip flowcytometry channel.

In addition, the present invention relates to methods for performingmultiplexed analysis. In particular, the methods herein employ one ormore fixative reagents to affix and retain nucleic acid targets withinthe cell, thereby allowing multiplexed detection of desired protein andnucleic acid targets within the same cell. Additional details follow.

Microfluidic Device

The present invention includes a microfluidic device configured toperform a multiplexed series of assays on a sample, to deliver one ormore reagents to designated chambers on the device in a controlledmanner, to hydrodynamically focus sample portions, and to opticallydetect target nucleic acids and proteins. All of these functionalitiescan be performed on-chip (i.e., within the device) and in an automatedmanner.

As seen in FIG. 1A, an exemplary microfluidic device 100 includes aplurality of assay chambers 121-123, 125, where each assay chamber isindividually addressable. The device 100 can include any useful n numberof chambers, where n can be of from 1 to about 5000. The assay chambers121-123, 125 are provided to contain a volume of a sample fluid havingtargets of interest (e.g., any target herein). As will be describedfurther below, labels, fixative reagents, amplification reagents,stimulants, stains, buffers, and other reagents, may be introduced tothe microfluidic device 100 and interact with the targets in the assaychambers 121-123, 125.

The device can include any useful type of chamber, e.g., an assaychamber configured to conduct a multiplexed series of assays; a samplechamber for receiving and/or storing a test sample; an incubationchamber for incubating a test sample or a portion thereof (i.e., asample portion); a reagent chamber configured to store or transport oneor more reagents; a wash chamber configured to maintain a sample portionfor one or more washes; a capture chamber configured to physicallycapture one or more sample portions on a wall of the chamber (e.g., awall treated with a capture reagent); a sterilization chamber containingone or more reagents to sterilize or disinfect the test sample; apermeabilization chamber containing one or more reagents to permeabilizea cell in the test sample; a fixation chamber containing one or morefixative reagents to fix a cell in the test sample; and/or a wastechamber for storing one or more by-products of the assay. Each of thesechambers can be interconnected by a valve, a port, and/or a channel thatcan optionally include a valve in its fluidic path. Alternatively, thesame chamber (e.g., the assay chamber) can be configured to performmultiple functions (e.g., capture, stimulate, fix, permeabilize, label,release, incubate, etc. a sample portion).

The chambers can have any useful dimension, geometry, and/orconfiguration. For instance, the chambers can have any usefulcross-sectional dimension, such as a length, depth, width, diameter,and/or major axis of from about 1 μm to about 1 cm (e.g., of from 1 μmto 1 mm, 1 μm to 500 μm, 1 μm to 300 μm, 10 μm to 500 μm, 10 μm to 300μm, 50 μm to 500 μm, 50 μm to 300 μm, 100 μm to 1 mm, or 100 μm to 500μm). In other embodiments, the length of the chambers (e.g.,microchannels) is of from 10 μm to about 10 cm (e.g., from 100 μm to 1cm). The chambers can have any useful geometry, such as a rectangular,square, circular, trapezoidal, U-shaped, etc. cross-section.Furthermore, the chamber can be configured as a well or a channelconnected to one or more other chambers.

In some embodiments, the width and depth of the chamber (e.g.,microchannel) are generally selected to obtain the desired flowcharacteristics in the chamber and provide sufficient volume for theamount of sample or particles to be received by the chamber. Generally,the length of the chamber (e.g., microchannel) is selected to routefluid the appropriate distance between ports and chambers, for example.While example dimensions of channels have been provided herein forreference, other dimensions may be used in other embodiments.

The device can include any useful type and number of structures (e.g.,chambers, channels, wells, ports, etc.) to perform the desiredmultiplexed reactions and/or to establish the desired fluidic pathway.FIG. 1A shows an exemplary fluidic network to effect a multiplexednetwork. The fluidic network is defined within a substrate 170 andincludes one or more assay chambers 121-123, 125 connected to a mainchannel 140. This main channel 140, in turn, is connected to a main port110. This fluidic layout is particularly beneficial to distribute thesame substance to all chambers. For instance, the test sample can bedelivered to the main port 110 and transported through the main channel140 to equally distribute or aliquot a test sample portion into eachassay chamber 121-123, 125. By maintaining an equal pressure differencebetween the main port 110 and the other ports 111-113, 115, an equalamount of the test sample can be distributed into each assay chamber121-123, 125.

The device can include one or more ports 111-113, 115 in fluidiccommunication with one or more assay chambers 121-123, 125. As can beseen, the first port 111 is most directly connected to the first assaychamber 121. In this fluidic network, opening and closing the first port111 (or the valve or fluidic connector that is connected to the firstport 111) can control flow and transport through the first chamber 121.For instance, by closing all ports and then opening the main port 110and the first port 111, a fluidic pathway is established between themain port 110 and the first port 111 (i.e., through the main channel 140and the first assay chamber 121) but all other fluidic pathways areclosed, thereby facilitating transport of a substance only to the firstassay chamber 121. In a similar manner, fluidic pathways and fluidtransport can be controlled between any of the chambers 121-123, 125 andports 110-113, 115, 160. For instance, the second port 112 is mostdirectly connected to the second assay chamber 122, the third port 113to the third assay chamber 123, and the n^(th) port 115 to the n^(th)chamber 125.

The device can include an on-chip flow cytometry module. In oneinstance, the on-chip flow cytometry module includes one or more flowcytometry channels facilitated to focus a desired sample portion througha detection area, as well as one or more sheath channels. As can be seenin FIG. 1A, the device can include a flow cytometry channel 155 that isconfigured to contain flow of a sheath fluid from first and secondsheath ports 151, 152. Connecting the sheath port 151, 152 to the flowcytometry channel 155 is the sheath channel 153, 154. Alternatively,only one sheath port can be present but configured to be in fluidiccommunication with two sheath channels, where one sheath channel isdisposed to the right of the flow cytometry channel and the other sheathchannel is disposed to the left of the flow cytometry channel. Anyuseful configuration of sheath port(s), sheath channel(s), and flowcytometry channel(s) can be employed. Additional details on the module,including excitation sources and detectors, are described herein.

The flow cytometry channel 155 is in fluidic communication with the mainchannel 140 and an exit port 160. In this manner, the main channel 140provides a fluidic pathway between each assay chamber 121-123, 125 andthe flow cytometry channel 155. As each assay chamber is individuallyaddressable, individual fluidic pathways can be established between theassay chamber and the flow cytometry channel, thereby allowing eachsample portion to be analyzed on an individual basis by the on-chip flowcytometry module.

The device can include one or more ports to provide fluidic access tothe microfluidic device. In particular, such ports can be in fluidiccommunication with a sample reservoir or a reagent reservoir in amanifold in order to deliver sample portions and reagents to the device(e.g., by way of one or more fluidic connections and/or connectors, asdescribed herein). Any useful number of ports may be present tofacilitate fluid access to and from the microfluidic device at desiredlocations. Each port may be useful as an inlet, an outlet, or acombination of inlets and outlets depending on the fluidic pathway beingestablished on-chip. The ports can have any useful dimension, such asfrom about 100 μm to about 2 (e.g., a cross-sectional dimension, such asa length, width, major axis, or diameter of about 500 μm).

The device may be fabricated from any useful material and employing anyuseful methodology. For instance, the substrate can include, e.g.,quartz, glass, polycarbonate, fused-silica, poly(dimethyl siloxane), apolymer, a metal, a semiconductor, or a transparent substrate, as wellas composites and multi-layered, laminated, or bonded forms thereof.Exemplary methods of fabrication include rapid prototyping,microfabrication (e.g., by casting, injection molding, compressionmolding, embossing, ablation, thin-film deposition, and/or ComputerNumerically Controlled (CNC) micromachining), photolithography, etchingtechniques (e.g., wet chemical etching, reactive ion etching,inductively coupled plasma deep silicon etching, laser ablation, or airabrasion techniques), methods for integrating these structures intohigh-throughput analysis equipment (e.g., integration with a microplatereader or a control instrument, such as a computer), methods forfabricating and integrating valves (e.g., one or more pneumatic valves),methods for integrating structures with a transducer array, methods formodifying surfaces (e.g., by including a layer of extracellular matrixcomponents, such as with a protein solution (e.g., Cell-Tak™, asdescribed herein), fibronectin (FN), laminin, Matrigel™, and/or RGDpeptide, or by including a layer of a globulin protein, such as albuminor an immunoglobulin), methods for including one or more capture arrays(e.g., a capture array including one or more capture agents provided ina high-density array on a substrate), and methods for providing vias orinlets (e.g., by piercing, drilling, ablating, or laser cutting), suchas those described in U.S. Pat. No. 8,257,964; and U.S. Pub. Nos.2012/0231976, 2012/0214189, 2011/0129850, 2009/0251155, and2009/0036324, each of which is incorporated herein by reference in itsentirety.

On-Chip Flow Cytometry Module

The present invention includes a device having an on-chip flow cytometrymodule. The module includes one or more structures to hydrodynamicallyfocus a sample portion. In addition, the module can be used with one ormore excitation sources and detectors to detect one or more detectablelabels.

As seen in FIG. 1A, the device 100 includes a flow cytometry channel 155located downstream of the assay chambers 121-123, 125. In use, a sampleportion containing labeled targets can be transported from the assaychamber towards the flow cytometry channel. A flow cytometry buffersolution (or sheath fluid) is then transported through the sheathchannel 153, 154 from the sheath port 151, 152. The sheath solutionhydrodynamically focuses the target (e.g., one or more labeled targetslocated on or within an intact cell from the test sample) as the flow ofsheath fluid meets the flow of sample fluid from the assays chambers.Additional ports may present to produce multidirectional and moreefficient hydrodynamic focusing of the target. The hydrodynamicallyfocused targets may then be analyzed by flow cytometry excitation energysources and detectors positioned to analyze particles in the flowcytometry channel 155.

The sheath fluid can be any useful fluid that does not detrimentallyaffect the sample portion. For instance, when the sample portionincludes one or more intact cells, then the sheath fluid can be anisotonic, buffered solution capable of maintaining cell viability orintegrity (e.g., any buffer herein, such as a phosphate buffered saline(PBS), a tris(hydroxymethyl) aminomethane (Tris) buffer, a Tris-bufferedsaline (TBS), a Tris/sucrose/ethylenediamine tetraacetic acid solution(TSE), etc.). In addition, the sheath fluid should be capable ofhydrodynamically focusing a sample portion. Throughput of the sampleportion through the flow cytometry channel may be related to the degreeof hydrodynamic focusing and size of the flow cytometry channel. In oneexample, a 10:1 focusing ratio can provide a throughput of around 100cells per minute. Additional methods of configuring appropriate flowrates to establish hydrodynamic are described herein, as well as in LiuP et al., “Microfluidic fluorescence in situ hybridization and flowcytometry (glowFISH),” Lab Chip 2011 Aug. 21; 11(16):2673-9; and PerroudT D et al., “Microfluidic-based cell sorting of Francisella tularensisinfected macrophages using optical forces,” Anal. Chem. 2008 Aug. 15;80(16):6365-72, each of which is incorporated herein by reference in itsentirety.

The on-chip flow cytometry module can be configured for use with anexcitation source and a detector to detect labels in hydrodynamicallyfocused samples. The excitation source and detector need not be locatedon-chip. For instance, the excitation source and detector can beexternal components that are configured to be used with samples that arehydrodynamically focused on-chip. In addition, the excitation source anddetector can be used to detect the sample portion in any usefulstructure of the device, such as within the assay chamber, within themain channel, and/or within the flow cytometry channel. In this manner,various multiplexed reactions can be monitored in any useful manner.

The excitation source and detector can be employed for any suitableimaging technique, e.g., confocal microscopy, differential interferencecontrast (DIC) microscopy, fluorescence microscopy, or combinationsthereof. Allowing the targets to be imaged within the same device usedto perform flow cytometry (i.e., imaged on-chip) may advantageouslyallow for both imaging and flow cytometry analysis to be performed on asame sample within a short period of time. For example, cells may beimaged within the assay chamber. The quantity, concentration, or both oflabeled components of the cells can be visually observed.

Generally, any number of excitation sources and detectors may be used,depending on the flow cytometry analysis, as will be understood in theart. The selection of energy source wavelength and detector sensitivitymay be based in part on the type of label(s) used, and the detectionmethodology. Any useful excitation source can be employed, such as alaser, a diode laser, an optical fiber, or any other optical source(e.g., a mercury or xenon lamp), which can be used in combination withone or more filters, mirrors, etc.

Detectors can be positioned to receive both the forward energy (e.g., inthe direction of the energy source illumination) and scatter energy(e.g., at an angle from the direction of energy source illumination), asis understood in the art of flow cytometry. Any type of optical energydetector may generally be used, such as a photomultiplier tube (PMT), acharge-coupled device (CCD), which can be used in combination with ananalog-to-digital converter, an amplifier, a filter cube, etc.

Additional device structures and components are described herein, aswell as in Liu P et al., Lab Chip 2011 Aug. 21; 11(16):2673-9; Perroud TD et al., Anal. Chem. 2008 Aug. 15; 80(16):6365-72; and Srivastava N etal., “Fully integrated microfluidic platform enabling automatedphosphoprofiling of macrophage response,” Anal. Chem. 2009 May 1;81(9):3261-9, as well as U.S. Pat. No. 7,999,937, each of which isincorporated herein by reference in its entirety.

Platform

The platform of the invention includes a microfluidic device (e.g., anyherein), as well as one or more components configured to provide one ormore samples or reagents on-chip (e.g., by use of a manifold, a pumpingsystem, and/or a controller) and to detect one or more labeled targets(e.g., by use of an excitation source, a detector, and/or a controller).

FIG. 1B provides an exemplary platform 1000 including a microfluidicdevice 1010, a manifold 1020, a pumping system 1030, and a controller1040. As can be seen, the device 1010 is in fluidic communication withthe manifold 1020. The pumping system 1030 is configured to transport areagent or a sample from the manifold 1020 and into the device 1010.Finally, a controller 1040 controls the manifold 1020, the pumpingsystem 1030, as well as any other component capable of beingelectronically controlled (e.g., one or more pressure sensors, pressurevalves, etc.). Each of these components is detailed below.

The manifold 1020 can include one or more reservoirs M1-M13 configuredto store one or more agents or fluids, such as samples, reagents, sheathfluid, waste, etc. Each reservoir includes an outlet configured tointerface with a connector 1080 (e.g., a fitting, a luer lock, aferrule, a nut, an o-ring, a Y-connector, a T-connector, etc.,optionally including a gasket and/or a frit) and/or a fluidic connection1070 (e.g., a tubing, optionally including an in-line filter, column,etc.). The connector provides a tight, leak-proof connection between thereservoir and the tubing, whereas the fluidic connection contains andtransports the agent to a port of the microfluidic device. In someembodiments, the diameter of the fluidic connection may be selected toreduce cell loss by axial dispersion in the tubing (e.g., a diameterless than about 500 μm) and to reduce cell adhesion (e.g., a PEEK tubingor a PTFE tubing). Connectors can be present on the device (at one ormore ports) and the manifold (at one or more outlets).

Any useful fluidic pathway can be adapted to effectively deliver agentsfrom the manifold to the device. For instance, the manifold 1020 caninclude a reservoir M1 configured to contain the sample and additionalreservoirs M4-M9 configured to contain a reagent (e.g., a protein label,a fixative reagent, a nucleic acid label, wash buffers, etc.). Each ofthese reservoirs can be connected to a fluidic connection 1070. Thesefluidic connections 1070 can be combined to form a main connection 1050,where the desired agent can be delivered on-chip by use of one or valves1061, 1062 (e.g., multi-port valves, an autosampler, etc.). In anotherinstance, the manifold 1020 can include a reservoir M2 configured tocontain a sheath fluid, which in turn is connected to the sheath channelof the device 1010 by way of a fluidic connection having a valve 1063.In yet another instance, the manifold 1020 can include a reservoir M3configured to collect the analyzed sample portions or waste that istransported from the exit port of the device by way of a fluidicconnection having a valve 1064.

A reservoir can be optionally connected to each port that is closest toan assay chamber. For example, the manifold can include a plurality ofreservoirs M10-M13, and each reservoir can be connected to a separateport of the device 1010 by way of a fluidic connection having a valve1065-1068. A skilled artisan would understand that any usefulconfiguration of fluidic connections can be employed to direct agentsfrom the manifold to the device, as well as any useful routines tocontrol pressure or flow of these agents through the fluidicconnections. For instance, the manifold 1020 can be coupled to a pumpingsystem 1030, which is then coupled to each reservoir and configured toindividually control pump rates, flow rates, displacement rates, and/orpressure of the agent through the device. The pumping system can includeone or more electronic pressure pumps coupled to an inert gas supply(e.g., a nitrogen gas supply) in order to generate pressure to beapplied by the electronic pressure pumps. In some examples, pressuresmay be applied up to about 5 psi. Higher or lower pressures may also beapplied.

One or more controllers can be employed to control the pumping system,the valves, etc. For instance, a controller 1040 can be coupled thepumping system 1030 and configured to control the pumping system 1030(e.g., by executing any useful pumping routine). For instance, thecontroller can be configured to fluidically deliver a particular agentinto a particular chamber to perform a multiplexed assay. The controllercan be configured to fluidically deliver an agent by applying a controlsignal to the appropriate valve to open or close that valve. By applyingsuch signals in an appropriate sequence, the desired fluidic pathway canbe established.

Using the device in FIG. 1A and the platform in FIG. 1B as an example,the controller 1040 can be configured to fluidically deliver a testsample portion from a first reservoir M1 to the main port 110 and thento each assay chamber 121-123, 125, thereby providing a sample portionin each assay chamber. The desired fluidic pathway (i.e., from the firstreservoir M1 to each assay chamber 121-123, 125) can be established byfirst providing a control signal to valves 1061, 1062 to open theconnection between the first reservoir M1 and the main connection 1050.As can be seen, this main connection 1050 is connected to the main port110 of the device 1010, 100. To establish flow to the assay chambers, apressure gradient should be generated between the main port 110 and theports 111-113, 115 located near the assay chambers. The pressuregradient can be established by the controller, e.g., by providing acontrol signal to valves 1065-1068 to open the connection between theports 111-113, 115 of the device 100 and the reservoirs M10-M13 in themanifold 1020. In this manner, the appropriate fluidic pathway has beenestablished (e.g., from reservoir M1; through the main connection 1050;to the main port 110; continuing through the main channel 140; into eachassay chamber 121-123, 125; out through each port 111-113, 115; and thenfinally into each holding reservoir M10-M13).

Fluid flow can begin by applying pressure to the appropriate reservoirM1 in the manifold, where this pressure will transport the sample inthat reservoir along the established fluidic pathway. The controller canbe programmed to apply pressure driven flow in this configuration for apredetermined amount of time (a sample loading time). In a similarmanner, other agents can be delivered from a reservoir to any desiredchamber(s) of the device.

The controller 1040 can be configured to provide one or more labels forthe desired target(s), such as by being configured to fluidicallydeliver a protein label from a second reservoir M4 to at least one assaychamber 121-123, 125, thereby providing a labeled sample portion;fluidically deliver a fixative reagent from a third reservoir M5 to atleast one assay chamber (e.g., chamber 121) containing the labeledsample portion, thereby providing a fixed sample portion; andfluidically deliver a nucleic acid label from a fourth reservoir M6 toat least one assay chamber (e.g., chamber 121) containing the fixedsample portion, thereby providing a multi-labeled sample portion.Finally, the controller 1040 can be configured for on-chip flowcytometry analysis, such as by being configured to fluidically deliverthe multi-labeled sample portion(s) from at least one assay chamber121-123, 125 to a flow cytometry channel 155; and fluidically deliverone or more sheath fluids from a fifth reservoir M2 to the sheath port151. In this manner, the controller can be configured to perform anyuseful routine for preparing, incubating, and detecting sample portions,where each individual, single cell is tested in a multiplexed manner.

The platform can include any other useful components. As seen in FIG.1C, the platform 1100 can include a device 1110 positioned on a stage1150, a manifold 1120 configured to deliver one or more agents, apumping system 1130 coupled to the manifold 1120, as well as anexcitation source 1160 and a detector 1170 aligned with a detection areaof the device (gray area in device 1110). The stage 1150 can optionallybe coupled to a stage controller 1159 (e.g., to control stage movement)and a temperature controller 1155 (e.g., to control a heater or coolerlocated on or near the stage). As described herein, the controller 1140can be configured to control the pumping system 1130 and can optionallyserve as an integration hub configured to control other components, suchas the excitation source 1160, the detector 1170, the stage controller1159, and the temperature controller 1155. The controller 1140, in turn,can be coupled to a processor 1180. The processor 1180 can be configuredto receive detection signals from the flow cytometry detectors and/or toprocess the detection signals, e.g., by fitting peaks to the detectionsignal, identifying peak locations, and/or generating populationhistograms. In this manner, the methods of the invention can beautomated by using such a platform.

FIG. 2A-2D provides schematics of an exemplary platform. Provided areschematics of the device (FIG. 2A-2B), the platform components (FIG.2C), and the optical laser pathway (FIG. 2D).

FIG. 3A-3C provides another exemplary platform. Provided is a schematicof a microfluidic device 300 having a main port 310 and an exit port 360in a substrate 370 (FIG. 3A) The device 300 includes a plurality ofchambers 321-330, where each chamber is in fluidic communication with aport 311-320. The flow cytometry channel 355 is in fluidic communicationwith the main channel 340. Sheath channels 356, 367 are located on eachside of the flow cytometry channel 355 and connected by one sheath port351, where the sheath channels 356, 367 are configured tohydrodynamically focus a sample portion in the flow cytometry channel355. As shown in the inset of FIG. 3A, the microphotograph shows thejunction between the sheath channels 356, 367 and the flow cytometrychannel 355 and the detection area (dashed circle) within the flowcytometry channel 355.

FIG. 3B shows the pumping scheme 3000 including a device 3010 that is influidic communication with fluidic connections having valves 3061-3064.Each fluidic connection, in turn, is coupled to a pressurized reservoirM31-M34 that is individually controlled by a pressure controller3031-3034. A processor 3080 and an integration hub 3040 can beconfigured to control the valves 3061-3064 and pressure controllers3031-3034.

FIG. 3C shows the detection scheme 3100 including a device 3110 alignedwith an optical fiber 3111 coupled to a forward scatter detector 3120(e.g., a PMT, such as to observe sample portions in the assay chamber).Also provided are components to detect fluorescence signals during flowcytometry analysis. These components include an objective 3130 alignedto the flow cytometry channel of the device 3110, an excitation source3160 (e.g., a laser source having the desired excitation wavelength,such as 488 nm), a filter cube 3175, and a plurality of detectors3171-3174 (e.g., PMTs configured to detect a particular wavelength orwavelength range).

The device and platform can include any other useful structures orcomponents, such as one or more pre-concentrator channels (e.g., asdescribed in U.S. Pat. No. 7,828,948, which is incorporated herein byreference in its entirety); optical tweezers (e.g., as described inPerroud T D et al., Anal. Chem. 2008 Aug. 15; 80(16):6365-72, which isincorporated herein by reference in its entirety); micropores (e.g., asdescribed in Perroud T D et al., “Isotropically etched radial microporefor cell concentration, immobilization, and picodroplet generation,” LabChip 2009 Feb. 21; 9(4):507-15, which is incorporated herein byreference in its entirety); LNA flow-FISH components (e.g., as describedin Wu M et al., “miRNA detection at single-cell resolution usingmicrofluidic LNA flow-FISH,” Methods Mol. Biol. 2014; 1211:245-60; Liu Pet al., Lab Chip 2011 Aug. 21; 11(16):2673-9; and Wu M et al.,“Single-cell protein analysis,” Curr. Opin. Biotechnol. 2012 February;23(1):83-8, each of which is incorporated herein by reference in itsentirety); separation/extraction components (e.g., filters, posts,membranes, weirs (optionally including beads), matrices, or high voltageelectrodes for performing on-chip capillary electrophoresisseparations); heating components (e.g., electrodes, resistive heaters,heated stages, or filaments); pumps (e.g., active or passive pumps, suchas an electric pump or a low flow rate peristaltic pump or applicationof negative pressure, such as by actuating a valve); a membrane (e.g.,placed within a channel and/or a chamber); a multifunctional sensor(e.g., to measure temperature, strain, and electrophysiological signals,such as by using amplified sensor electrodes that incorporate siliconmetal oxide semiconductor field effect transistors (MOSFETs), a feedbackresistor, and a sensor electrode in any useful design, such as afilamentary serpentine design); a microscale light-emitting diode (LEDs,such as for optical characterization of the test sample); anactive/passive circuit element (e.g., such as transistors, diodes, andresistors); an actuator; a wireless power coil; a device for radiofrequency (RF) communications (e.g., such as high-frequency inductors,capacitors, oscillators, and antennae); a resistance-based temperaturesensor; a photodetector; a photovoltaic cell; a diode; one or morecomponents to operate a transducer, such as a power source to operate anelectrode; a data-processing circuit powered by the power source andelectrically connected to the transducer (e.g., a counter electrode, areference electrode, and at least one said working electrode); and/orone or more components for autonomous remote monitoring of a sample,such as an analog-to-digital converter, a radiofrequency module, and/ora telemetry unit (e.g., configured to receive processed data from adata-processing circuit electrically connected to the detectioncomponent and to transmit the data wirelessly).

Methods for Performing Multiplexed Analysis

The present invention also includes methods for performing multiplexedanalysis. In particular embodiments, the method includes use of a deviceand/or a platform described herein. In yet other embodiments, themethods herein allow for single-cell analysis of a sample portion whileminimizing sample consumption and reducing complicated, manual fluidhandling procedures. Rather, the methods herein employ a microfluidicdevice to contain and control reactions of small volumes (e.g.,nanoliter- or picoliter-scale volumes) and an automated platform toexecute fluid handling protocols.

FIG. 9 provides an exemplary method 900 for performing multiplexedanalysis in a microfluidic device. In a first step 901, a test sampleportion is loaded into the device. To aliquot test sample into eachassay chamber of the device, the test sample portion can be loaded intoa main port (e.g., in fluidic communication with a main channel, whichin turn is in fluidic communication with each assay chamber).

In a second step 902, the test sample portion is captured within eachassay chamber. For instance, flow is employed in the device to deliveragents to the desired chamber. If the test sample was not captured, thenflow could displace the test sample outside of the assay chamber. Thus,in the exemplary methods herein, the test sample is captured on asurface of the assay chamber for the duration of the multiplexed assay,and then the multiplexed-labeled sample portion is released fordetection and/or analysis. Any useful capture reagent (e.g., such as anyherein) can be used to capture the test sample. In one embodiment, atissue adhesive (e.g., Cell-Tak™, a commercially available adhesivecontaining polyphenolic proteins extracted from Mytilus edulis) can beused to coat a chamber surface, and a protein (e.g., a protease) can beused to cleave proteins within the tissue adhesive in order to releasethe captured cells.

The next optional steps can be employed for sample preparation purposes.For instance, one optional step 903 can include culturing a sampleportion, e.g., in order to increase the cell number to a particularconfluence.

Another optional step 904 can include stimulating a sample portion witha stimulant (e.g., an exogenous molecule, peptide, protein, nucleicacid, etc., such as any herein). For instance, complicated reactionnetworks within cells can generally be perturbed by a stimulant, and thebiomolecular components of these networks can be identified by exposinga cell with that stimulant and performing multiplexed analyses fornumerous possible targets. For instance, ionomycin is a potent calciumionophore, and calcium signaling is a general signaling pathway forvarious cellular processes. In T cells, ionomycin mediates T cellactivation and, thus, serves as a useful stimulant for studying T cellactivation. Other exemplary stimulants include one or more signalingmolecules, modulators, activators, inhibitors (e.g., kinase inhibitors,such as wortmannin or LY294002; mitotic inhibitors, such as vincristine;and phosphatase inhibitors, such as okadaic acid), ionophores (e.g.,calcium ionophores, such as ionomycin), endotoxins (e.g.,lipopolysaccharide (LPS)), lipids (e.g., glycolipids), tumor promoters(e.g., phorbols and phorbol esters, such as12-O-tetradecanoylphorbol-13-acetate (TPA) orphorbol-12-myristate-13-acetate (PMA)), antineoplastic agents (e.g.,nocodazole), antitumor agents (e.g., paclitaxel), antibiotics (e.g.,brefeldin A (BFA)), antibodies, drugs, hormones, toxins (e.g.,phalloidin), etc.

Yet another optional step 905 includes treating the sample portion witha fixative reagent and, optionally, washing the fixed sample. Inparticular for whole cell-based analysis, preservation of cellularstructures, morphology, surface characteristics, and internalbiomolecules can be important. Thus, one or more fixative reagents canbe employed to prevent autolysis. In addition, different fixativereagents can be employed to stabilize different structures within thecell sample. Any useful fixative reagent (e.g., any herein) can beemployed.

In a next step 906, the method includes incubating the sample portion inan assay chamber with a protein label. As described herein, in certainsteps, the incubating step 906 is performed on captured samples.Furthermore, if the multiplexed assay includes more than one proteinlabel, then step 906 includes incubating with a first protein label(including a mixture of the first protein label with another label,e.g., a second protein label configured to detect a second targetprotein). Each protein label is configured to detect particular targetprotein(s) (e.g., any herein), thereby providing a labeled sampleportion.

Various optional steps may be conducted with a labeled sample portion(e.g., from step 906 or any following steps). In one optional step 907,the labeled sample portion is treated with a fixative reagent and,optionally, washed. In another optional step 908, the sample portion ispermeabilized, e.g., with a permeabilization reagent (e.g., anydescribed herein). When the sample portion includes cells, thenpermeabilization may be needed for a label to penetrate cellularmembranes.

If more than one protein labels are employed for the assay, then themethod can optionally include a step 909 of incubating the sampleportion in an assay chamber with a second protein label, where thesecond protein label is configured to detect a second target protein(e.g., any herein), thereby providing a doubly-labeled sample portion.Again, after this incubating step 909, the method can optionally includea step 910 of treatment with a fixative reagent and with an optionalwash reagent (e.g., any buffer, such as any herein).

In yet another step 911, the method includes incubating the sampleportion in an assay chamber with a nucleic acid label. Furthermore, ifthe multiplexed assay includes more than one nucleic acid label, thenstep 911 includes incubating with a first nucleic acid label (includinga mixture of the first nucleic acid label with another label, e.g., asecond nucleic acid label configured to detect a second target nucleicacid). Each nucleic acid label is configured to detect particular targetnucleic acid(s) (e.g., any herein), thereby providing amultiplexed-labeled sample portion. Optionally, the nucleic acid labelcan include a signal that can be amplified. For instance, if the nucleicacid label includes an amplifiable region (e.g., an amplifiable nucleicacid portion), then one or more amplification reagents can be used toform numerous amplicons based on the amplifiable region, therebyproviding an amplified signal. When the amplifiable region is present ona circular template, then the amplification reagents can be used to forma concatemer having numerous amplicon portions. If those amplicons canthen be bound to a detectable marker, then the amplified signal providesan amplified detectable signal.

FIG. 4A provides one exemplary method of amplifying a nucleic acidsignal. As can be seen, the method includes the use of various labels todetect the target, such as a nucleic acid label 410 that binds to thetarget 401; a secondary label 420 that binds to the nucleic acid label410; a tertiary label 431, 432 that binds to the secondary label 420;and a quaternary label 441, 442 that binds to the tertiary label 431,432. Each of these labels is discussed below.

In general, the nucleic acid label 410 includes a nucleic acid-bindingregion 411 configured to detect the target nucleic acid 401. The nucleicacid label 410 can include one or more other chemical components, suchas a detectable marker (e.g., any herein, such as a fluorophore, aquantum dot, etc.). Alternatively, the nucleic acid label can includeone or more affinity agents to allow binding of the nucleic acid labelto other secondary, tertiary, quaternary, etc. labels that facilitatedetection, amplification, in a specific and/or selective manner. Forinstance, the nucleic acid label 410 can include one or more affinityagents 412, 413 having an optional linking region that covalently bindsan affinity agent 412, 413 to the binding region 411.

The affinity agent can be one partner of a conjugating pair. As anexample only, the conjugating pair can include a first affinity agentbeing a protein (e.g., digoxigenin or DIG) and a second affinity agentbeing an antibody that specifically binds that protein (e.g., ananti-digoxigenin antibody or anti-DIG antibody). Another exemplaryconjugating pair includes biotin (first affinity agent) and streptavidin(second affinity agent). Other conjugating pairs and affinity agentsforming that pair are described herein. As seen in FIG. 4A, the nucleicacid label 410 includes two affinity agents 412, 413 flanking thebinding region 411. The number of affinity agents for each nucleic acidlabel can be selected to improve selectivity and/or sensitivity. Inaddition, the same or different affinity agents can be present for eachnucleic acid label.

When the construct of the nucleic acid label includes a first affinityagent of a conjugating pair, then the secondary label can include asecond affinity agent of the conjugating pair. In this manner, thesecondary label specifically binds to the nucleic acid label (or theprimary label). As seen in FIG. 4A, the secondary label 420 includes asecond affinity agent (A2) that binds to the first affinity agent (A1)of the primary nucleic acid label 410. For instance, A1 can be DIG, andA2 can be an anti-DIG antibody. The secondary label can include adetectable marker (e.g., any herein). Alternatively, the secondary labelcan be configured to bind to a tertiary label.

As seen in FIG. 4A, two tertiary labels 431, 432 are employed for eachsecondary label 420, where each tertiary label binds to the secondarylabel. Thus, a tertiary label can include a third binding partner thatbinds to the second partner (e.g., as seen in FIG. 4A, the tertiarylabel 431 includes a third binding partner (labeled with +) that bindsto the second partner A2 420). In one example, the first tertiary labelis a first proximity ligation probe 431 having a third binding partner(e.g., an antibody, such as an anti-mouse antibody) configured to bindto the second binding partner A2 420 (e.g., a mouse anti-DIG antibodyconfigured to bind to DIG in the primary nucleic acid label). The firstproximity ligation probe 431 also includes a first nucleic acidsequence, and the second proximity ligation probe 432 includes a secondnucleic acid sequence and a fourth binding partner (e.g., labeled with adetectable marker, where the third and fourth binding partners can bethe same or different).

The proximity ligation probes provide an extended nucleotide, which inturn serves as a binding site for a quaternary label capable of beingligated and amplified. For instance, the quaternary label can be apadlock probe (e.g., a single-stranded nucleic acid sequence configuredto bind to both the first and second nucleic acid sequences of the firstand second proximity ligation probes) or two connector probes (e.g., twosingle-stranded nucleic acid sequences, where a first connector probesequence is configured to bind to a portion of the first proximityligation probe and the second connector probe sequence is configured tobind to a portion of the second proximity ligation probe). As seen inFIG. 4A, the first connector probe 441 includes a nucleic acid sequencehaving a portion that binds to a terminus of the first proximityligation probe 431 and another portion that forms the circular template(i.e., once the first and second connector probes are ligated with aligase). The second connector probe 442 includes a nucleic acid sequencehaving a portion that binds to the second proximity ligation probe 432and another portion that forms the circular template. Once the first andsecond connector probes 441, 442 are ligated (e.g., using a DNA or RNAligase), then a circular template is formed. Then, by including one ormore enzymes (e.g., polymerases, such as a DNA or RNA polymerase),optional primers, nucleotides, etc., a concatemer is generated based onthe circular template. Finally, if needed, one or more detectablemarkers capable of binding a sequence in the concatemer can be included.Additional modifications and methods for amplification and for probedesign are provided in U.S. Pat. Nos. 5,665,539, 6,511,809, 6,558,928,6,878,515, 7,074,564, and 8,268,554, as well as U.S. Pub. No.2014/0194311, each of which is incorporated herein by reference in itsentirety.

Finally, returning to FIG. 9, the method 900 includes the step 913 ofperforming an on-chip flow cytometry assay within the device. Tofacilitate hydrodynamic focusing of captured sample portion, the methodcan include detaching the sample portion (e.g., the multiplexed-labeledsample portion), thereby providing a detached sample portion; anddelivering the detached sample portion to the flow cytometry channel.The step 913 can include establishing flow within the flow cytometrychannel in order to provide a hydrodynamically focused sample portion(e.g., including the multiplexed-labeled sample portion from at leastone assay chamber), activating an excitation source (e.g., therebyexciting the target or target particles with a flow cytometry excitationsource), and receiving energy emitted from the labeled targets with aflow cytometry detector

FIG. 10 provides an alternative exemplary method 1000 including a fixingstep after use of a protein label but prior to treatment with a nucleicacid label. Such a method can be useful, e.g., for using a fixativereagent that cross-links a target nucleic acid to retains its locationwithin the cell. In particular, when the fixative reagent detrimentallyinteracts with a protein label, then the protein label can include oneor more inert detectable markers (e.g., quantum dots, instead of aprotein-based fluorophore, which can be destroyed by one or morefixative reagents, e.g., EDC).

Accordingly, in FIG. 10, the method 1000 is one for performingmultiplexed analysis with at least two protein labels and at least onenucleic acid label. The method 1000 requires a first step 1001 ofloading a test sample into the device; a second step 1002 of capturingthe test sample (e.g., within each assay chamber); a third step 1006 ofincubating the sample portion with a first protein label; a fourth step1109 of incubating the sample portion with a second protein label; afifth step 1010 of treating the sample portion with a fixative reagent(e.g., a cross-linking agent, such as EDC or DCC) and an optional washreagent (e.g., a buffer or any wash described herein); a sixth step 1011of incubating the sample portion with a first nucleic acid label; and afinal step 1013 of performing an on-chip flow cytometry assay. Regardingthe fifth step 1010, FIG. 4B shows an exemplary schematic of a fixativereagent (e.g., EDC) that cross-links the miRNA target with a protein toform a cross-linked miRNA-protein complex.

The method 1000 includes various optional steps, such as step 1003 ofculturing a sample portion; step 1004 of stimulating a sample portionwith a stimulant; step 1005 of treating the sample portion with afixative reagent and, optionally, washing the fixed sample; step 1007 oftreating the sample portion with a fixative reagent and, optionally,washing the fixed sample, where the fixative reagent of steps 1005,1007, 1010 can be the same or different; step 1008 of permeabilizing thesample portion with a permeabilization reagent (e.g., any describedherein); and/or step 1012 of amplifying a signal of the nucleic acidlabel.

FIG. 11 provides an alternative exemplary method 1100 including twofixing steps and a permeabilizing step. This method can be useful, e.g.,for labeling cell surface proteins and then later using a permeabilizingreagent for labeling intercellular protein and/or nucleic acid targets.

Accordingly, in FIG. 11, the method 1100 is one for performingmultiplexed analysis with at least one cell surface protein label, atleast one intracellular protein label, and at least one nucleic acidlabel. The method 1100 requires a first step 1101 of loading a testsample into the device; a second step 1102 of capturing the test sample(e.g., within each assay chamber); a third step 1105 of treating thesample portion with a fixative reagent (e.g., paraformaldehyde, oranother chemical fixative described herein); a fourth step 1106 ofincubating the sample portion with a first protein label configured todetect a cell surface protein; a fifth step 1108 of permeabilizing thesample portion with a permeabilization reagent (e.g., Triton™ X-100 orany described herein); a sixth step 1109 of incubating the sampleportion with a second protein label configured to detect anintracellular protein; a seventh step 1110 of treating the sampleportion with a fixative reagent (e.g., a cross-linking agent, such asEDC or DCC); an eighth step 1111 of incubating the sample portion with afirst nucleic acid label configured to detect miRNA and/or mRNA; a ninthstep 1112 of amplifying a signal of the nucleic acid label(s); and afinal step 1113 of performing an on-chip flow cytometry assay.

The method 1100 includes various optional steps, such as step 1103 ofculturing a sample portion; step 1104 of stimulating a sample portionwith a stimulant; and step 1107 of treating the sample portion with afixative reagent and, optionally, washing the fixed sample, where thefixative reagent of steps 1105, 1107, 1110 can be the same or different.

Multiplexed Analysis of Targets

The devices, platforms, and methods of the inventions can be employed toperform multiplexed analysis of any target nucleic acid, target protein,target post-translational modification (PTM), and/or other targetbiomolecules, as well as any combinations of these.

Exemplary target nucleic acids include mRNA, miRNA, RNA (e.g., anyherein), DNA (e.g., any herein), including single-stranded formsthereof, double-stranded forms thereof, sense and antisense formsthereof, as well as any having natural or non-natural nucleobases ornucleic acids. Exemplary target proteins include one or more cellsurface proteins, transmembrane or membrane proteins, intercellularproteins, cytosolic proteins, nuclear pore complexes, enzymes,structural proteins (e.g., fibrous proteins), globular proteins,hormones, etc. Exemplary target post-translational modification:phosphorylation, adenylylation, dephosphorylation, glycosylation,ubiquitination, acylation, alkylation (e.g., methylation or ethylation),amidation, deamidation, carbamylation, carboxylation, hydroxylation,nitrosylation, succinylation, sulfation, glycation, myristoylation,palmitoylation, prenylation, glypiation, addition of one or morecofactors (e.g., a lipoate, flavin, heme, or phosphopantetheinylmoiety), etc. Exemplary target biomolecules include one or morecarbohydrates, lipids, glycosaminoglycans, steroids, etc.

For instance, the devices, platforms, and methods of the inventions canbe employed to analyze miRNA (see, e.g., FIG. 5A-5D), miRNA with a cellsurface protein (e.g., CD69, such as in FIG. 6A-6C), and a portfolio oftargets, such as any combination of mRNAs, miRNAs, cell surfaceproteins, phosphorylation, cytosolic proteins, and glycosylation (see,e.g., FIG. 8A-8F). Any useful cellular process, such as those in FIG. 7,can be studied using the multiplexed process described herein.

Labels for Target Detection

For multiplexed detection, the present invention employs labels that areselective for the desired target. For instance, a protein label is anagent that is configured to detect a target protein, and a nucleic acidlabel is an agent that is configured to detect a target nucleic acid,and a PTM label is an agent that is configured to detect a targetpost-translational modification. Exemplary labels are described herein.

In one example, the protein label includes a protein-binding region andoptionally, a detectable marker (e.g., any herein). The protein-bindingregion can include any useful protein-binding region (e.g., an antibody,as well as fragments thereof). In addition, the protein label can beused in combination with a secondary protein label, in which thesecondary label binds to the protein label. For instance, the proteinlabel can include a protein-binding region that binds to the target anda first affinity agent (e.g., biotin of a conjugating pair includingbiotin and avidin); and the secondary label can include a secondaffinity agent (e.g., avidin) and an optional detectable marker. In thismanner, a plurality of labels can be employed to facilitate selectivedetection of the target and sensitive observation of any detectablesignals. In some embodiments, a secondary label is configured todirectly or indirectly bind to the protein label (e.g., therebyproviding an excited label for the multi-labeled sample portion).

In another example, the nucleic acid label includes a nucleicacid-binding region (e.g., with sufficiently complementary tospecifically bind a target nucleic acid) and an affinity agent (e.g.,such as a first partner of a conjugating pair). Additional secondary,tertiary, quaternary, etc. labels (e.g., as described herein) can beemployed with the primary nucleic acid label (see, e.g., FIG. 4A andassociated text). In some embodiments, a secondary label is configuredto directly or indirectly bind to the nucleic acid label (e.g., therebyproviding an excited label for the multi-labeled sample portion).

In yet another example, the PTM label includes a region configured toselectively bind to a post-translational modification or apost-translation, modified protein. For instance, the label candifferentially bind to a protein having a post-translationalmodification, as compared to a non-modified protein (e.g., an antibodyselective for a phosphorylated protein, as compared to its non-modifiedtype; a lectin affinity agent that selectively binds sugar moieties; ora cationic carbocyanine dye that preferentially stains highly acidicproteins (e.g., phosphoproteins and calcium-binding proteins, such as1-ethyl-2-[3-(3-ethylnaphtho[1,2d]thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1,2d]thiazoliumbromide)).

In another instance, the PTM can include a moiety capable of beingreactive or being activated, where the reactive/active moiety can thenbe reacted with a detectable marker (e.g., a glycol moiety on aglycosylated protein, where this moiety can be oxidized to an aldehyde,which in turn can be reacted with a detectable marker to form a labeled,detectable conjugate). Any useful detectable marker can be employed,such as a fluorescent hydrazide (e.g., a Pro-Q® Emerald 300 dye) to forma fluorescently detectable conjugate, a digoxigenin hydrazide to form aconjugate that can be bound by an antidigoxigenin antibody having adetectable marker (e.g., a fluorophore, quantum dot, or detectableenzyme), and/or a biotin hydrazide to form a conjugated that can bebound by a streptavidin-containing moiety (e.g., horseradish peroxidaseor alkaline phosphatase conjugates of streptavidin). Additional PTMlabels and methods for PTM detection are described in Steinberg T H etal., “Rapid and simple single nanogram detection of glycoproteins inpolyacrylamide gels and on electroblots,” Proteomics 2001 July;1(7):841-55; and Steinberg T H et al., “Global quantitativephosphoprotein analysis using Multiplexed Proteomics technology,”Proteomics 2003 July; 3(7):1128-44, each of which is incorporated hereinby reference in its entirety. In addition, commercially available labelsinclude, e.g., Pro-Q® Diamond Phosphoprotein Gel Stain (P-33300,Molecular Probes®, available from Life Technologies, Grand Island, N.Y.,Thermo Fisher Scientific Inc.); and Pro-Q® Emerald 300 or 488Glycoprotein Gel Stain Kit (P-21855 or P-21875, Molecular Probes®,available from Life Technologies).

For the labels, any useful affinity agent can be employed. In certainembodiments, the first and second affinity agent bind together with ahigh binding affinity (e.g., at least about 10⁻⁴ M, usually at leastabout 10⁻⁶ M or higher, e.g., 10⁻⁹ M or higher) that is sufficient toensure specificity and/or sensitivity.

Exemplary affinity agents include an antibody, such as polyclonal,monoclonal, and single chain forms thereof, fragments thereof,polyepitopic specific forms thereof, multispecific forms thereof,chimeric forms thereof, and humanized forms thereof; a receptor; alectin; an aptamer (e.g., a nucleic acid aptamer); a chemical moiety; asmall molecule (e.g., a cyclical organic compound); a solublecell-surface receptor or derivative thereof; an antibody mimetic (e.g.,an affibody); a cofactor (e.g., biotin); a coenzyme; an enzyme; a sugarmoiety; a polysaccharide; a lipid; a toxin; a click-chemistry moiety(e.g., an azido group, an alkynyl group, a dienophile group, or a dienegroup); a steroid (e.g., digoxigenin); and/or a protein (e.g., avidin,streptavidin, or neutravidin).

The affinity agent can be one partner of a conjugating pair. Exemplaryconjugating pairs include an antibody and an antigen for that antibody;an aptamer and an affinity agent selected to specifically bind thataptamer, such as an anti-thrombin aptamer and thrombin; a lectin and asugar moiety, such as concanavalin A and a glycoprotein; digoxigenin(DIG) and an anti-DIG antibody; a mouse antibody and an anti-mouseantibody; a hapten and an anti-hapten antibody; biotin and streptavidin;fluorescein and an anti-fluorescein antibody optionally labeled with adetectable marker (e.g., an enzyme); an optionally substituted alkynylgroup and an optionally substituted azido group; an optionallysubstituted diene having a 4 π-electron system and an optionallysubstituted dienophile or an optionally substituted heterodienophilehaving a 2 π-electron system; a nucleophile and a strained heterocyclylelectrophile; an optionally substituted amino group and an aldehyde or aketone group; an optionally substituted amino group and a carboxylicacid group; an optionally substituted hydrazine and an aldehyde or aketone group; an optionally substituted hydroxylamine and an aldehyde ora ketone group; and/or a nucleophile and an optionally substituted alkylhalide. Any other useful conjugating pair may be employed. In addition,each affinity agent can be optionally labeled with a detectable marker(e.g., an enzyme, a fluorophore, a quantum dot, etc., or any herein) byway of an optional linking agent (e.g., an poly(ethylene glycol), analkylene group, etc.). Additional affinity agents and labels aredescribed in Wu M et al., Curr. Opin. Biotechnol. 2012 February;23(1):83-8; and Boyce M et al., “Bringing chemistry to life,” Nat.Methods 2011 August; 8(8):638-42, each of which is incorporated hereinby reference in its entirety.

Furthermore, affinity agents can be selected based on overlappingselectivity between conjugating pairs. For instance, a primary label caninclude a DIG as an affinity agent, and the secondary label can includea mouse anti-DIG antibody, which binds to DIG. The tertiary label, inturn, can include an anti-mouse antibody, which binds to the mouseanti-DIG antibody. In this manner, sets of affinity agents, not justpairs, can be designed and implemented in primary, secondary, tertiary,quaternary, etc. labels.

Detectable Marker

The probes and labels herein can include any useful detectable marker.Exemplary detectable markers include a dye, such as an electroactivedetection agent, a fluorescent dye, a luminescent dye, achemiluminescent dye, a colorimetric dye, a radioactive agent, etc.; aparticle, such as a microparticle, a nanoparticle, a latex bead, acolloidal particle, a magnetic particle, a fluorescent particle, etc.; atag (e.g., an electroactive tag, an electrocatalytic tag, a fluorescenttag, a colorimetric tag, a quantum dot, a nanoparticle, a microparticle,a barcode, a radio tag (e.g., an RF tag or barcode); an affinity agent(e.g., any herein, such as avidin or biotin); an enzyme that canoptionally include one or more linking agents and/or one or more dyes;an enzyme that can be detected by way of an enzymatically cleavablesubstrate (e.g., horseradish peroxidase by way of a cleavable substratehaving an oxidizable group, thereby providing an optically detectablesignal; or alkaline phosphatase by way of a cleavable substrate having aphosphate group); an amplifying agent (e.g., a PCR agent, such as apolymerase, one or more deoxyribonucleotide triphosphates, a divalentmetal (e.g., MgCl₂), a template DNA, and/or a primer (e.g., for bindingto a selective region of the target nucleic acid); a globulin protein(e.g., bovine serum albumin); a sandwich assay reagent; and/or acatalyst (e.g., that reacts with one or more substrates to provide adetectable signal). Additional detectable markers are described in Wu Met al., Curr. Opin. Biotechnol. 2012 February; 23(1):83-8; and Boyce Met al., Nat. Methods 2011 August; 8(8):638-42, each of which isincorporated herein by reference in its entirety.

Capture Reagents

As described herein, the devices and methods of the invention caninclude capturing a sample portion on a surface of the device (e.g.,within an assay chamber, such as on at least one chamber wall). In oneembodiment, a solution is employed to deposit one or more adhesionlayers within a chamber or channel of the device. The adhesion layer canbe formed from any useful substance, such as a protein (e.g., anextracellular matrix protein, a globulin protein, a structural protein,or a fibrous protein, including albumin, an immunoglobulin, fibrin,collagen, fibronectin, laminin, entactin, or tenascin C), a polypeptide,a protein fraction, a proteinaceous mixture (e.g., Cell-Tak™, acommercially available tissue adhesive containing polyphenolic proteinsextracted from Mytilus edulis and formulated in 5% acetic acid; orMatrigel™, a gelatinous protein mixture from Engelbreth-Holm-Swarm mousesarcoma cells), a polymer (e.g., a charged polymer, such as cationicpoly-L-lysine (PLL) or polyethyleneimine (PEI)), and/or a peptiderecognition motif (e.g., a Arg-Gly-Asp motif).

When a protein component is employed, then any useful enzyme or chemicalagent can be used to release the captured sample portion. Exemplaryenzymes and chemical agents include Arg-C proteinase, Asp-Nendopeptidase, BNPS-Skatole, a caspase (e.g., caspase 1, 2, 3, 4, 5, 6,7, 8, 9 or 10), chymotrypsin, clostripain (clostridiopeptidase B),cyanogen bromide (CNBr), enterokinase, Factor Xa, formic acid, glutamylendopeptidase, granzyme B, hydroxylamine (e.g., NH₂OH), iodosobenzoicacid, LysC lysyl endopeptidase (Achromobacter proteinase I), LysNpeptidyl-Lys metalloendopeptidase, neutrophil elastase,2-nitro-5-thiocyanobenzoic acid (NTCB optionally with nickel), pepsin,proline-endopeptidase, proteinase K, staphylococcal peptidase I, tobaccoetch virus protease, thermolysin, thrombin, and/or trypsin.

Amplification

In some embodiments, the method includes use of one or more labelsincluding a nucleic acid sequence (e.g., a template, including a padlockor a circular template) capable of being amplified to provide anamplicon (e.g., short or long nucleic acid sequences, which includesconcatemers). In particular embodiments, the amplicon is produced by anamplification reaction, e.g., an isothermal amplification or a rollingcircle amplification. The amplifying step can include use of apolymerase (e.g., a DNA polymerase).

In yet other embodiments, the amplifying step includes performing anamplification reaction by providing one or more antibodies configured tobind to the primary nucleic acid label, one or more proximity ligationprobes configured to bind to the one or more antibodies, connectorprobes configured to form a circular template (e.g., wherecircularization is mediated by the use of one or more ligases) and tobind the proximity ligation probes, one or more enzymes (e.g.,polymerases) configured to generate a concatemer based on the circulartemplate, one or more nucleotides (e.g., dNTPs), and/or one or moresplint oligonucleotides (e.g., to bind the circular template and/or tobind to a terminus on each of the two connector probes)

Fixative Reagents

The fixative reagent can include any useful agent or compound configuredto form a bond (e.g., a covalent bond) between two reactive groups(e.g., a carboxyl group and an amino group or a phospho group and anamino group). Exemplary fixative reagents include a chemical fixative(e.g., formaldehyde, paraformaldehyde, glutaraldehyde, formalin,acetone, isopropanol, ethanol, and/or methanol) or a cross-linker, aswell as combinations thereof. Exemplary cross-linkers include those forforming a covalent bond between a carboxyl group (e.g., —CO₂H) and anamino group (e.g., —NH₂) or between a phospho group (e.g., —P(O)(OH)₂)and an amino group (e.g., —NH₂), such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) anddicyclohexylcarbodiimide (DCC), optionally used withN-hydroxysuccinimide (NHS) and/or N-hydroxysulfosuccinimide (sulfo-NHS).Other cross-linkers include those for forming a covalent bond between anamino group (e.g., —NH₂) and a thymine moiety, such assuccinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB); a hydroxyl group(e.g., —OH) and a sulfhydryl group (e.g., for a cysteine moiety), suchas p-maleimidophenyl isocyanate (PMPI); between an amino group (e.g.,—NH₂) and a sulfhydryl group (e.g., for a cysteine moiety), such assuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) and/or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); and between asulfhydryl group (e.g., for a cysteine moiety) and a carbonyl group(e.g., an aldehyde group, such as for an oxidized glycoproteincarbohydrate), such as N-beta-maleimidopropionic acidhydrazide-trifluoroacetic acid salt (BMPH) and/or3-(2-pyridyldithio)propionyl hydrazide (PDPH).

Permeabilization Reagents

The permeabilization reagents can include any useful agent or compoundconfigured to permeabilize cell membranes, or portions thereof.Exemplary permeabilization reagents include a surfactant, such asTriton™ X-100 (e.g., polyethylene glycolp-(1,1,3,3-tetramethylbutyl)-phenyl ether), sodium dodecyl sulfate(SDS), Tergitol-type NP-40 (nonyl phenoxypolyethoxylethanol), andpolysorbate 20 (Tween 20); an alcohol, such as methanol; a solvent(e.g., acetone or acetic acid); a glycoside, such as saponin ordigitonin; a protease, such as proteinase K; or an exotoxin, such asstreptolysin O.

Samples

Any useful test sample can be analyzed. Exemplary test samples caninclude one or more cells, including rare cells (e.g., primary cells,stem cells, cancer cells, etc.); a biopsy sample; a cell; a tissue; afluid; a swab; a biological sample (e.g., blood, serum, plasma, saliva,etc.); an environmental sample; a microorganism; a microbe; a virus; abacterium; a fungus; a parasite; a helminth; a protozoon; a nucleic acid(e.g., oligonucleotides, polynucleotides, nucleotides, nucleosides,molecules of DNA, or molecules of RNA, including a chromosome, aplasmid, a viral genome, a primer, or a gene); a protein (e.g., aglycoprotein, a metalloprotein, an enzyme, a prion, a kinase, a membraneprotein, a receptor, or an immunoglobulin); a metabolite; a cytokine; acofactor; a factor (e.g., a transcription factor); a sugar or saccharide(e.g., including polysaccharides and monosaccharides); a lipid; alipopolysaccharide; a salt; an ion; and/or a particle (e.g., cells,including macrophage cells, and beads, including beads having cells orother biological materials attached to the beads). Such samples can betested for any use, such as point-of-care cell-based assays, diagnosesof complex disorders, medical treatment (e.g., by tracking patientresponses to drug therapies), and/or multiplexed cellular analysis ofsignaling events on a single-cell level.

EXAMPLES Example 1: Microfluidic Platform for the Multiplexed Detectionof miRNA, mRNA, and Proteins at Single-Cell Resolution

MicroRNAs (miRNAs) are non-coding, small single-stranded RNAs thatmodulate and regulate gene expression in numerous biological systemsranging from cell development and differentiation, in immune responseand inflammation, and in pathological states, such as cancer andautoimmune diseases (see, e.g., Alvarez-Garcia I et al., “MicroRNAfunctions in animal development and human disease,” Development 2005;132:4653-62; Baltimore D et al., “MicroRNAs: new regulators of immunecell development and function,” Nat. Immunol. 2008; 9:839-45; Lodish H Fet al., “Micromanagement of the immune system by microRNAs,” Nat. Rev.Immunol. 2008; 8:120-30; Kasinski A L et al., “Epigenetics and genetics.MicroRNAs en route to the clinic: progress in validating and targetingmicroRNAs for cancer therapy,” Nat. Rev. Cancer 2011; 11:849-64; andAlevizos I et al., “MicroRNAs in Sjogren's syndrome as a prototypicautoimmune disease,” Autoimmun. Rev. 2010; 9:618-21). As a result,miRNAs have become the subject of intense research as potentialdiagnostic disease biomarkers as well as therapeutic targets for drugdevelopment.

While traditional bulk assay methods (e.g., microarray, RT-qPCR, andsequencing technologies) have yielded high volume of informationregarding the expression profiles of miRNAs, the progress towardsdeveloping miRNA clinical diagnostics and therapies have been slow, duelarge in part to the complex nature of miRNA biology. The expression andfunction of miRNAs are tissue-specific—the same miRNA can exert oppositemodulatory effects on signaling pathways, modulate entirely differentsignaling pathways, or have no effect, all depending on the tissue typeand cellular context.

Without the ability to monitor mRNAs, proteins, and transient signalingevents along with miRNAs in the same cell, it is very difficult toascertain what relationship, if any, miRNA expression has to health ordisease of that cell. Bulk profiling methods generate averaged miRNAmeasurement from heterogeneous cell populations, and the informationregarding how miRNA expression levels relate to mRNA and proteinindicators of the cellular physiological state is lost in the samplepreparation process. In addition, averaged cellular signal from apopulation can mask the cell-to-cell variability of response within thatpopulation, and therefore single-cell resolution analysis of miRNAlevels will yield information otherwise unattainable using bulk methods.

To address the need for a new technology that provides single-cellresolution analysis of miRNAs in relation to mRNA and proteinbiomarkers, we have developed an automated, microfluidic platform withaccompanying molecular assays that enable rapid processing of intactcells (˜8 hours) to simultaneously detect small non-coding RNA (e.g.,miRNAs), mRNAs, proteins, and post-translational modifications atsingle-cell resolution, with >95% reduction in sample and reagentrequirement (see, e.g., Wu M et al., “Single cell microRNA analysisusing microfluidic flow cytometry,” PLoS One 2013; 8(1):e55044 (6pages); Wu M et al., “Microfluidic molecular assay platform for thedetection of miRNAs, mRNAs, proteins, and posttranslationalmodifications at single-cell resolution,” J. Lab. Autom. 2014 December;19(6):587-92; Wu M et al., “Microfluidically-unified cell culture,sample preparation, imaging and flow cytometry for measurement of cellsignaling pathways with single cell resolution,” Lab Chip 2012 Aug. 21;12(16):2823-31; and Srivastava N et al., “Fully integrated microfluidicplatform enabling automated phosphoprofiling of macrophage response,”Anal. Chem. 2009 May 1; 81(9):3261-9). The device provides fluorescentimages, as well as flow cytometry measurements of each target orbiomarker, and provides an unprecedented, comprehensive look into themolecular physiological state of single cells.

At the heart of the platform is a glass microfluidic chip with tenindividually addressable cell holding chambers or assay chambers (FIG.2A), thereby allowing ten different conditions per experiment. Eachdevice is fluidically connected to fourteen programmable valves, reagentreservoirs, and pumps (FIG. 2C) that use positive pressure to drive themovement of cells and reagents on and off the chip. A series of customadd-on features including the pumps and valves, pressure controllers,and temperature control are controlled by the user using a graphic userinterface (GUI) with programmable features for automated samplepreparation. For instance, the in-house designed software allows theexperimenter to control the pressure, temperature, and valves byprogramming each step of the experiment to run automatically. The chip'sconfiguration allows for automated interrogation of ten different celltypes or experimental conditions, using only 270 nL of reagent perchamber, thereby reducing the reagent cost more than 95%.

The microfluidic chip sits in a manifold retrofitted to an invertedfluorescent microscope with an attached camera (FIG. 2B), and the usercan visually track fluid and cell movement during the experiment, aswell as perform bright field and fluorescent microscopy analysis afterautomated sample preparation (FIG. 2C). After sample preparation andimage capture, the chip and manifold are moved to a custom-builtmicro-flow cytometer (FIG. 2D). Cells are detached by proteolyticcleavage (e.g., employing a protease) and hydrodynamically focused atthe center of the chip for on-chip flow cytometry. The optical fiber ispositioned on top of the chip, and aligned to the hydrodynamicallyfocused path of the cells. The laser is applied from the bottom of thechip, and the signal from the passing cells is recorded by one or moredetectors (e.g., photomultiplier tubes, charge-coupled devices, etc.)situated underneath the chip.

In order to detect miRNAs at single-cell resolution, a novel flowcytometry compatible fluorescent in situ hybridization (flow-FISH) assayto detect miRNAs using locked nucleic acid (LNA) containing probes hasbeen developed. The LNA flow-FISH assay combines the advantage of LNA'shigh melting temperature with the specificity of proximal ligation androlling circle amplification to provide highly specific amplification ofotherwise undetectable miRNA signals in an intact cell (FIG. 4A), whichthen can be visualized via microscopy and quantified using flowcytometry (see, e.g., FIG. 6A-6C).

The miRNA can be detected in any useful manner. Similar to the nucleicacid label in FIG. 4A, a double digoxigenin (DIG) labeled LNA-containingprobe can be hybridized to the target, a mature miR155. The nucleic acidportion of the probe allows for specific hybridization to the target,whereas the affinity agents (e.g., DIG) allows the probe to be capturedby one or more other affinity agents, such an antibody (e.g., ananti-DIG antibody).

The subsequent steps allow for further probes to be bound to the DIG-LNAprobe and to form a circular template. For instance, a monoclonalanti-DIG antibody can be bound to the DIG affinity agent. Then, a pairof proximity ligation probes is bound to the monoclonal anti-DIGantibody, where the proximity ligation probe includes a secondaryantibody configured to bind to the anti-DIG antibody and anoligonucleotide region. Next, connector probes were provided, in whichthe connector probe includes a nucleic sequence portion that iscomplementary to a terminal portion of the proximity ligation probes. Inuse, the connector probes are hybridized to a terminal portion of theproximity ligation probe, and then ligated to form a circular templatecontaining a short recognition sequence.

Finally, rolling circle amplification of the circular template isperformed to yield single-stranded concatemers. For fluorescencedetection, the concatemer can be hybridized to a labeled detection probethat is complementary to a recognition sequence encoded by the circulartemplate. The labeled detection probe can include a detectable label(e.g., any herein) and a nucleic acid sequence that is complementary toa recognition sequence, or a portion thereof.

The detection of miRNA species by LNA-flow FISH can be multiplexed withmRNA detection, and the isothermal nature of rolling circleamplification makes multiplexing with protein immunostaining possible.The multiplexing of miRNA and protein detection using LNA flow-FISH wasdemonstrated using miR155 and T-cell activation marker CD69 protein inactivated T cells, and the flow-FISH results show comparable sensitivityto qPCR (see Example 2 herein).

Other molecular assays that profile cell surface protein markers,transient cell surface receptor activation, intracellular proteins,post-translational modifications (e.g., phosphorylation and/or dynamicglycosylation of proteins), and mRNA have also been developed for themicrofluidic platform (see, e.g., FIG. 8A-8F and Example 4 herein). Allmicrofluidic molecular assays developed on the platform are compatiblefor multiplexing with each other, thereby providing a systems-levelanalysis of miRNAs in the native cellular context at a single-cellresolution. The platform and accompanying assays will advance theknowledge of miRNA function and their correlation with disease states,and holds great potential for both basic miRNA research and thedevelopment of multiplexed miRNA/mRNA/protein biomarker panels fordisease diagnostics and companion diagnostics.

Example 2: Single-Cell microRNA Analysis Using Microfluidic FlowCytometry

MicroRNAs (miRNAs) have cell type and cell context-dependent expressionand function. miRNAs function by directly binding the 3′ untranslatedregions (UTRs) of target mRNAs and recruit the RNA-induced silencingcomplex to degrade target mRNA (see, e.g., Krutzfeldt J et al.,“MicroRNAs: a new class of regulatory genes affecting metabolism,” Cell.Metab. 2006; 4:9-12). In humans, over 1000 miRNAs have been identified(see, e.g., Griffiths-Jones S, “The microRNA registry,” Nucleic AcidsRes. 2004; 32:D109-11), and each miRNA can potentially repress hundredsof target mRNAs, indicating the importance and complexity of this generegulation system.

Several methods have been developed for detection of miRNAs: Northernblotting, oligonucleotide microarrays, quantitative PCR (qPCR) assays,next generation sequencing, and in situ hybridization (ISH) (see, e.g.,Valoczi A et al., “Sensitive and specific detection of microRNAs bynorthern blot analysis using LNA-modified oligonucleotide probes,”Nucleic Acids Res. 2004; 32:e175; Castoldi M et al., “A sensitive arrayfor microRNA expression profiling (miChip) based on locked nucleic acids(LNA),” RNA 2006; 12:913-20; Thomson J M et al., “A custom microarrayplatform for analysis of microRNA gene expression,” Nat. Methods 2004;1:47-53; Duncan D D et al., “Absolute quantitation of microRNAs with aPCR-based assay,” Anal. Biochem. 2006; 359:268-70; Raymond C K et al.,“Simple, quantitative primer-extension PCR assay for direct monitoringof microRNAs and short-interfering RNAs,” RNA 2005; 11:1737-44; Xu G etal., “Transcriptome and targetome analysis in MIR155 expressing cellsusing RNA-seq,” RNA 2010; 16:1610-22; and Nielsen B S, “MicroRNA in situhybridization,” Methods Mol. Biol. 2012; 822:67-84).

With the exception of qPCR and ISH, all of these methods require lysisand homogenization of cells in order to provide measurement of miRNAaveraged over a large number of cells. Cells are heterogeneous in natureand hence, in many applications, it is desirable to measure miRNA insingle cells. The advent of locked nucleic acid (LNA) containing probeshas enabled RT-qPCR and in situ hybridization (ISH) analysis of miRNA atsingle-cell resolution (see, e.g., Tang F et al., “MicroRNA expressionprofiling of single whole embryonic stem cells,” Nucleic Acids Res.2006; 34:e9; Tang F et al., “220-plex microRNA expression profile of asingle cell,” Nat. Protoc. 2006; 1:1154-9; Pena J T et al., “miRNA insitu hybridization in formaldehyde and EDC-fixed tissues,” Nat. Methods2009; 6:139-41; and de Planell-Saguer M et al., “Rapid in situcodetection of noncoding RNAs and proteins in cells and formalin-fixedparaffin-embedded tissue sections without protease treatment,” Nat.Protoc. 2010; 5:1061-73).

Single-cell miRNA RT-qPCR, however, requires many steps includingisolation of single cells followed by lysis, RNA extraction, andamplification. In addition, this technology has limited throughput.LNA-ISH allows detection of endogenous miRNA in single cells withoutlysis and RNA extraction, but it is labor-intensive, at times poorlyreproducible, and provides only a qualitative assessment. Thus, LNA-ISHis used most frequently for fixed tissue sections.

Microfluidic devices have attracted significant attention in single-cellanalysis (see, e.g., Wu M et al., “Single-cell protein analysis,” Curr.Opin. Biotechnol. 2012; 23:83-8; Yilmaz S et al., “Single cell genomesequencing,” Curr. Opin. Biotechnol. 2012; 23:437-43; and Powell A A etal., “Single cell profiling of circulating tumor cells: transcriptionalheterogeneity and diversity from breast cancer cell lines,” PLoS One2012; 7:e33788). We have developed microfluidic single-cell analysissystems including microfluidic bacterial rRNA flow-FISH and cellsignaling pathway profiling (Liu P et al., “Microfluidic fluorescence insitu hybridization and flow cytometry (muFlowFISH),” Lab Chip 2011;11:2673-9; and Wu M et al., “Microfluidically-unified cell culture,sample preparation, imaging and flow cytometry for measurement of cellsignaling pathways with single cell resolution,” Lab Chip 2012;12:2823-31). Here, we discuss a novel ten-chamber microfluidic chipplatform for multiplexed detection of miRNA and proteins in single cellsunder ten different experimental conditions simultaneously.

To study miRNAs at single-cell resolution, we have developed a novelmicrofluidic approach, where flow fluorescent in situ hybridization(flow-FISH) using locked-nucleic acid (LNA) probes is combined withrolling circle amplification to detect the presence and localization ofmiRNA. Instead of using tyramide signal amplification to visualize themiRNA-LNA probe duplex, we used rolling circle amplification (RCA) ofthe target miRNA signal to achieve robust and reliable signalamplification. The RCA amplification reagent has been previously used todetect proteins both in lysates and in cells (Gullberg M et al.,“Cytokine detection by antibody-based proximity ligation,” Proc. Nat'lAcad. Sci. USA 2004; 101:8420-4; and Leuchowius K J et al., “In situproximity ligation assay for microscopy and flow cytometry,” Curr.Protoc. Cytom. 2011; Chapter 9:Unit 9.36). In addition, RCA has a limitof detection between 10⁻¹⁴ to 10⁻¹³ molar (Gustafsdottir S M et al.,“Proximity ligation assays for sensitive and specific protein analyses,”Anal. Biochem. 2005; 345:2-9), providing the necessary sensitivity fordetection of miRNA in single cells.

Furthermore, our flow cytometry approach allows analysis ofgene-products potentially targeted by miRNA together with the miRNA inthe same cells. Thus, an added benefit of LNA-Flow FISH is thepossibility of multiplexing with protein immunostaining in the samecell. As described herein, we multiplexed miRNA detection withimmunostaining of a protein to show the multiplexing capability.

We demonstrate our method in Jurkat cells, a model cell line for thestudy of T cell activation (Weiss A et al., “The role of T3 surfacemolecules in the activation of human T cells: a two-stimulus requirementfor IL 2 production reflects events occurring at a pre-translationallevel,” J. Immunol. 1984; 133:123-8). 12-O-tetradecanoylphorbol13-acetate (PMA) and ionomycin were employed to trigger T cellactivation, which leads to production of transmembrane glycoprotein CD69and up-regulation of miR155 (Marzio R et al., “CD69 and regulation ofthe immune function,” Immunopharmacol. Immunotoxicol. 1999; 21:565-82;and Lu L F et al., “Foxp3-dependent microRNA155 confers competitivefitness to regulatory T cells by targeting SOCS1 protein,” Immunity2009; 30:80-91).

We visualized and quantified PMA- and ionomycin-induced CD69 and miR155in Jurkat cells using both microscopy and flow cytometry, demonstratingsimultaneous detection of miRNA and a target protein in the same cell.CD69 is a lectin C-type protein that is involved in T celldifferentiation through the Jak3/Stat5 pathway, and is the earliestinducible surface protein indicative of T cell activation (Martin P etal., “CD69 association with Jak3/Stat5 proteins regulates Th17 celldifferentiation,” Mol. Cell. Biol. 2010; 30:4877-89). miR155up-regulation in T cells is implicated in the negative regulation ofSOCS1 protein, which leads to increased levels of interleukin-2, acytokine necessary for T cell proliferation (Lu L F et al., Immunity2009; 30:80-91). The flow-FISH method herein was completed in ˜tenhours, utilizes only 170 nL of reagent per experimental condition, andis the first to directly detect miRNA in single cells using flowcytometry.

Materials and Methods

Microfluidic chip design and platform setup: The ten-chambermicrofluidic chip was designed in-house using AutoCAD 2010 (AutodeskInc., San Rafael, Calif.), photomasks were generated at Photo Sciences(Torrance, Calif.), and quartz microfluidic devices were fabricated byCaliper Life Sciences (Hopkinton, Mass.). An array of fourteen holes(500 μm in diameter, seven on each side) provided for fluid inlets andoutlets. Each serpentine chamber was individually addressable to allowten different conditions per experiment. The ten-chamber chip wassituated in a manifold with 14 pumps and valves (FIG. 2B-2C) that usedpositive pressure to drive the movement of cells and reagents on and offthe chip. The chip and manifold were placed in a custom micro-flowcytometer for flow cytometric analysis (FIG. 2D).

The device in this study contained ten horizontal fluidically isolatablemicrochannels with the following dimensions: width of 200 μm, depth of30 μm, and length of 72 mm, with each holding from 500 to 2000 cells and170 nL to 220 nL of fluid volume. Additional steps in chip packaging anddetails of the chip platform are described in, e.g., Wu M et al., LabChip 2012 Aug. 21; 12(16):2823-31.

Cell culture and stimulation: Jurkat cells were purchased from ATCC(TIB-152) and cultured in RPMI media (11875-093; Invitrogen, Carlsbad,Calif.) containing 10% FBS (100-500; Gemini, and 0.5 mg/ml penicillinand streptomycin (Ser. No. 15/240,062; Invitrogen). For stimulation ofJurkat cells, cells were seeded at 1×10⁶/ml for 0, 8, 16, 20, or 24hours with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (P8139; Sigma)and 1 μM ionomycin (13909; Sigma). After stimulation, the Jurkat cellswere fixed with 8% paraformaldehyde (PFA, Electron Microscopy Sciences,Hatfield, Pa.) in phosphate-buffered saline (PBS) for 10 min. Fixedcells were pelleted at 300 g for 5 min. and washed twice with PBS. Ifneeded, after stimulation, 5×10⁵ cells from each condition can be setaside for RNA extraction and RT-qPCR.

RT-qPCR: 5×10⁵ cells from each condition were set aside prior tofixation and pelleted at 300 g for 5 min. and washed twice with PBS.Total RNA was extracted using the RNeasy kit from Qiagen according tomanufacturer's instructions. The extracted RNA was quantified using aNanodrop 2000, and 100 ng of RNA was used to generate cDNA using themiScript II RT kit (Qiagen). All steps were performed according tomanufacturer's instructions. 100 ng of cDNA from each condition wassubsequently used in miScript hsa-mir-155 primer assay (Sangeraccession: MI0000681, Qiagen), normalized to positive control SNORD61(Qiagen). Expression level of miRNA155 was analyzed using the 2-ΔΔCtmethod. SNORD61 served as a positive control for normalization. miR155level at 0 h. served as basal level, and miR155 in PMA and ionomycintreated samples are expressed as fold changes compared with 0 hours.

Microchip surface treatment with Cell-Tak™ to reversibly capturenonadherent cells: The planar microfluidic cell preparation chip in thisstudy contained ten fluidically-isolatable chambers. The ten-channelmicrochip was cleaned with 10% bleach in filtered, deionized (DI) waterfor 15 min., followed by flushing with DI water to wash off all residualbleach. The working Cell-Tak™ solution (354240; BD Biosciences, SanJose, Calif.) was prepared by combining 15 μL of Cell-Tak™ with 575 μLof 0.1 M sodium bicarbonate (pH 8.0), followed by adding 10 μL of 1 NNaOH immediately prior to adsorption onto the chip. Freshly made workingCell-Tak™ solution was continuously driven into all ten-cell holdingchambers of the device for at least 15 min. to thinly coat themicrochannels with Cell-Tak™, followed by PBS wash for 5 min. to removeexcess Cell-Tak™ from the microchannels. The coated devices were usedwithin one day. Fixed Jurkat cells were introduced into the Cell-Tak™coated chip and captured on the microchannel surface for ISH.

On-chip LNA in situ Hybridization: The miR155 probe had the followingsequence: /5DigN/ACCCCTATCACGATTAGCATTAA/3Dig_N/(SEQ ID NO:1), and thescrambled probe had the following sequence:/5DigN/GTGTAACACGTCTATACGCCCA/3Dig_N/(SEQ ID NO:2). These LNA doubleDIG-labeled probes were purchased from Exiqon A/S, Vedbaek, Denmark.Fixed Jurkat cells were loaded into the chip, and allowed to settle andadhere to the microchannel surface for 30 min. During the settling time,the following solutions were freshly prepared: Solution 1 (0.13 M1-methylimidazole, 300 mM NaCl, pH 8.0, adjusted with HCl), Solution 2(0.16 M EDC in solution 1, adjusted to pH 8.0); and hybridization buffer(50% formamide, 2×SSC, 50 μg/ml yeast tRNA, 50 μg/ml salmon sperm DNA,50 mM NaPi). To permeabilize the Jurkat cells, 0.25% Triton™ X-100 inTBS was flown into all chambers for 10 min., followed by a 5 min. washwith solution 1. After incubation with Solution 1, Solution 2 was flowninto all chambers and cells were incubated for 20 min. at roomtemperature (RT), followed by a 5 min. wash with TBS. The cells werethen pre-hybridized for 30 min. at 62° C. in hybridization buffer(pre-warmed to 65° C.). All LNA probes were used at 10 pmol/25 μlhybridization buffer. Hybridization of LNA probes was performed at 80°C. for 90 seconds (s.), followed by 90 min. at 62° C. Following LNAprobe hybridization, all chambers were washed with 2×SSC with 50%formamide at 65° C. for 10 min. (flow 5 min., stop 5 min.), then washedwith 1×SSC for 20 min. (flow 5 min., stop 15 min.) at RT, and finallywashed with 0.1×SSC for 20 min. (flow 5 min., stop 15 min.) at RT.

Signal amplification using rolling circle amplification: The FITCDuolink® anti-mouse PLUS probe (92001-0030), Duolink® anti-mouse MINUS(92004-0030) probe, and Duolink® detection kit (92014-0030) from OlinkBiosciences AB (Uppsala, Sweden) were used to perform rolling circleamplification of miRNA signals as previously described (Wu M et al.,PLoS One 2013; 8(1):e55044).

After in situ hybridization (ISH) with DIG-labeled LNA probes, the cellswere blocked with 2% bovine serum albumin (BSA) for 30 min. at 37° C.and incubated with anti-DIG antibody (11333062910; Roche, Indianapolis,Ind.) at 1:50 for 1 h. at 37° C. Cells were then washed with TBS with0.05% Tween 20 (TBST) for 5 min. The detection of the miRNA/LNA probeduplex was accomplished by amplifying the anti-DIG antibody bound to theDIG labels on the LNA probes. The Duolink® anti-mouse PLUS and MINUSprobes were diluted 1:5 (20 μL PLUS, 20 μL MINUS, and 60 μL dilutionbuffer from kit) and incubated with cells for 1 h. at 37° C. Afterincubation, all chambers were washed for 5 min. with TBST, and thesubsequent ligation and amplification steps were done according to themanufacturer's instructions by using only one reaction volume for allten chambers. After on-chip sample preparation, cells were detached viaproteolytic cleavage using 100 μg/ml elastase (I.U.B.: 3.4.21.36,Worthington) and hydrodynamically focused for on-chip flow cytometry.

CD69 protein immunostaining multiplexed with LNA Flow-FISH: To multiplexprotein immunostaining with LNA flow-FISH, Jurkat cells were stainedwith anti-CD69-biotin antibody at 1:100 (13-0699-80, eBioscience) in PBSfor 30 min. at RT prior to permeabilization with 0.25% Triton™ X-100. Asolution of CD69 antibody was flown into all chambers, flow was thenstopped for 30 min. for incubation. All chambers were washed with TBSwith 0.05% Tween for 5 min., followed by 30 min. incubation with a 1 nMsolution of Qdot® 705-streptavidin conjugate (Q10161MP, Invitrogen) inPBS. Following Qdot® 70-streptavidin incubation, all chambers werewashed with TBST for 5 min. All chambers were then washed with TBST for5 min. The in situ hybridization procedure continues from this point onat the permeabilization step.

Microscopy and image analysis: Prior to imaging, the cells wereincubated with Hoechst stain (33342, Pierce) in PBS for 10 min.,followed by a 10 min. wash in PBS. Epi-fluorescent images were capturedat 60× magnification on an Olympus IX-71 microscope equipped with GFP,Texas Red, DAPI filters and Hamamatsu ORCA-R2 cooled CCD cameracontrolled via free micro-manager software. Images were artificiallycolored and overlaid in ImageJ.

On-chip laser induced fluorescence and flow cytometry: A 20-mW diodepumped solid-state laser at 488 nm (85-BCF-020-112; CVI Melles Griot,Carlsbad, Calif.) in an epifluorescence configuration was used forexcitation (see Wu M et al., PLoS One 2013; 8(1):e55044). The laser beamwas reflected and focused upon the detection region using a long passdichroic mirror (LPD01-488S; Semrock, Rochester, N.Y.) and an asphericlens (5722-H-B; New Focus, Santa Clara, Calif.), respectively. Forwardscattering was collected and channeled to the active area of aphotomultiplier tube (PMT) based detector (H5784-20; Hamamatsu,Bridgewater, N.J.) using a custom-made sculpted tip silica optical fiber(1000 μm core, 2000 μm sculpted spherical tip (Polymicro Technologies,Phoenix, Ariz.). Laser-induced fluorescence emission was first collectedvia the same aspheric lens used for focusing and subsequently passed onfor detection via the dichroic mirror used in the excitation leg of theapparatus as described above.

For multiplexed detection, an eight channel Hamamatsu linear multi-anode(LMA) PMT coupled with filter optics (H9797TM; Hamamatsu, Bridgewater,N.J.) was used. Only four channels of the LMA PMT were used in thisconfiguration, and the filtering was selected for green, yellow, red,and far-red fluorescence detection. The green fluorescence was detectedusing longpass dichroic mirror (DMT560: Hamamatsu, Bridgewater, N.J.)and a bandpass filter (BPF534_30). For yellow fluorescence detection inthe second channel, longpass dichroic mirror (DMT650) and bandpassfilter (BPF585_40). Red fluorescence was detected third channel of theLMA PMT via a longpass dichroic (DMT740) and filtered using a thirdbandpass filter (BPF692_40). Finally, far-red fluorescence detection wasaccomplished by cascading the remaining florescence signal onto thefourth LMA PMT channel using a mirror, and filtered onto that channelsdetection region using a fourth bandpass filter (BPF785_62).

Data acquisition was performed for all five photomultiplier voltages(488 nm scatter, green, yellow, red, and far red) by a data acquisitionmodule (NI USB-6259; National Instruments, Austin, Tex.). In-housesoftware for data acquisition and recording was scripted using LabVIEW,and the data were further analyzed and processed using a custom “PeakFinder” application also scripted using LabVIEW. The Peak Finderapplication fit the peak of the raw voltage signals from the PMT with apolynomial fit and generated the peak amplitude and width values.

Results and Discussion

Microfluidic platform and assay design: A ten-chamber microfluidic chipwas designed for sample preparation and on-chip flow cytometry (FIG. 3A,left). The chip had fluidically-isolatable chambers, each capable ofholding up to 2000 cells. The microchannel surfaces were pre-coated withCell-Tak™ solution to facilitate capture of non-adherent Jurkat cellsfor the on-chip hybridization and immunostaining. After samplepreparation, the cells in the chambers are detached using proteolyticcleavage, and driven by positive pressure to the center of the chipwhere they are hydrodynamically focused (FIG. 3A, right) andinterrogated using micro-flow cytometry using a custom built setup (FIG.2D).

Our microfluidic platform's ultra-low reagent consumption substantiallyreduces the cost of LNA flow-FISH (˜100-fold reduction from ˜$150/sampleto <$1.50/sample). The flow-FISH assay uses a novel combination of LNAprobes with RCA signal amplification for a robust miRNA signal that canbe quantified by flow cytometry as well as visualized by microscopy.

A schematic depicting the miRNA LNA Flow-FISH method is shown in FIG.4A. Once the cells are loaded into the chambers and captured byCell-Tak™, miR155 LNA probe with digoxigenin (DIG) conjugated to both 5′and 3′ termini was hybridized to mature intracellular miR155. After theLNA probe hybridizes with miR155, monoclonal anti-DIG antibodies(anti-DIG mAb) bound the DIG labels at both ends of the LNA probe. Apair of antibody/oligonucleotide probes (one positive and one negativeproximity ligation probes) was used to bind to the anti-DIG antibodies.After binding to the anti-DIG antibody, the two antibody/oligonucleotideprobes were ligated with two additional oligonucleotides (connectorprobes) to form a circular template for the subsequent rolling circleamplification with Phi29 bacterial polymerase. FITC-labeledoligonucleotide detection probes complementary to the ligated circulartemplate were hybridized with the resultant circular concatemers. EachRCA amplified LNA-probe/miRNA duplex became visible as fluorescent dots(FIG. 6A) and can also be detected by flow cytometry. RCA amplificationprovided improved signal specificity because it occurs only when acircular template is generated, and the detection of the circularproduct was accomplished using sequence-specific hybridization probes.

LNA Flow-FISH analysis of PMA and ionomycin induced miR155 upregulation:To track the expression of PMA and ionomycin induced miR155 over time,Jurkat cells were stimulated with PMA and ionomycin (for 0, 8, 16, 20,and 24 h.) and then loaded in duplicate into the ten chamber chip (FIG.5A). The top five chambers were hybridized with double DIG-labeledmiR155 LNA probe, and the bottom five chambers were hybridized withdouble DIG labeled random scrambled probe as negative control (labeled“SC” in FIG. 5A).

The RCA-amplified miR155 fluorescence was quantified using on-chip flowcytometry, and the fluorescence histograms were overlaid (FIG. 5B). Thenormalized median fluorescence values from three separate experimentswere plotted as percent of maximal fluorescence from each sample (FIG.5C).

Both the overlay in FIG. 5B and the bar graph of median values in FIG.5C showed an incremental increase in miR155 for Jurkat cells understimulation with PMA and ionomycin. Hybridization to the randomscrambled probe produced low level of background fluorescence thatremained constant throughout the time course, and the background wassubtracted from the miR155 measurements. The miRNA flow-FISH resultswere verified using population RT-qPCR analysis and calculated relativefold change from 0 h. (FIG. 5D).

Both flow-FISH and qPCR showed that miR155 increased significantly from0 h. at 16 h., 20 h., and 24 h., with p values <0.01; miR155 increasedfrom 0 to 8 h., but the change was not statistically significant(p>0.1). The upregulation of miR155 by PMA and ionomycin detected by ourLNA flow-FISH method corroborated the existing findings of miR155upregulation in activated T cells and not in resting T cells (Thai T Het al., “Regulation of the germinal center response by microRNA-155,”Science 2007; 316:604-8).

Multiplexed analysis of CD69 protein expression and miR155 upregulation:The high melting temperature of LNA probes provides superior specificityand rapid hybridization for detecting small miRNAs, but the highhybridization temperature can reverse formaldehyde fixation, and miRNAscan be washed away. A second fixation step (FIG. 4B) using1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) was performedsubsequent to formaldehyde fixation to irreversibly cross-link the miRNAto the neighboring amino acid residues (see, e.g., Pena J T et al., Nat.Methods 2009; 6:139-41). As seen in FIG. 4B, the 5′-phosphate of miRNAwas activated by EDC to form an intermediate, which was cross-linked toneighboring amino groups of proteins. The fixation with EDC allowedirreversible cross-linking that tethered miRNAs inside the cells duringthe high temperature hybridization conditions with LNA containingprobes. The EDC fixation step retained miRNAs inside the cell butdestroyed protein epitopes and protein based fluorophores, such asphycoerythrin.

To solve this problem, we used quantum dot labeled secondary antibodyfor multiplexed protein detection with LNA flow-FISH because quantumdots are inert to EDC fixation. Immunostaining with anti-CD69 antibodyand Qdot® 705-labeled secondary antibody was performed prior to fixationby EDC. At time 0, no CD69 and almost no miR155 can be seen. The sizeand intensity of green miR155 dots increase as duration of stimulationincreases. After microscopy, the cells were detached and analyzed usingflow cytometry (FIG. 6A), the median fluorescence values from miR155 andCD69 are plotted in the same graph (FIG. 6B), showing increase of bothmiR155 and CD69 under PMA and ionomycin stimulation. Significant CD69induction was detected 8 h. post stimulation. miR155 upregulationoccurred later than CD69 protein induction, detectable starting at 16h., indicating that signaling events surrounding CD69 protein inductionwas activated earlier by PMA and ionomycin than those that lead tomiR155 up-regulation.

CONCLUSION

We have developed a novel flow-FISH method for measuring relative miRNAchanges at single-cell resolution using a ten-chamber microfluidic chipplatform. The biggest advantage of the flow-FISH method is thecapability to multiplex the detection of miRNAs with proteinimmunostaining in the same cell, and preserve the cell-to-cellheterogeneity within the population (FIG. 6C). While we have showndetection of a single protein, it is possible to detect multipleproteins in the same cell using antibodies labeled with differentfluorophores or quantum dots. This multiplexing capability opens up manypotential applications for miRNA flow-FISH in both clinical and basicbiological sciences. One potential clinical application is the profilingof miRNA levels in complex clinical samples such as peripheral bloodmononuclear cells (PBMCs) from patients with leukemia or autoimmunedisorders. The expression level of up to ten different miRNA in tenimmune subsets can be analyzed in one microfluidic chip experiment.miRNA expression levels in many different cell types can be assessed andtracked as biomarkers indicative of disease progression or response totherapy. If miRNA copy number quantitation is required for developmentof a specific clinical diagnostic assay, custom flow-FISH quantitationbeads can be developed by conjugating known numbers of synthetic miRNAsonto polystyrene beads. The calibration beads can be captured on thechip, hybridized to the same LNA FISH probe, and amplified on the chipalong with experimental samples under the same conditions to provide astandard curve from which the miRNA copy number per cell can bedetermined.

For application in basic science, miRNA flow-FISH can be used to developfunctional assays for the discovery of miRNA targets in vivo. One miRNAand dozens of its putative mRNA and protein targets can besimultaneously quantified using a combination of LNA flow-FISH formiRNAs, traditional flow-FISH for the mRNAs, and immunostaining for theproteins. Time course experiments tracking the miRNA and targetmRNA/protein levels in the presence of miRNA antagonists or mimics canvalidate mRNA and protein targets in vivo. Finally, since we havealready successfully demonstrated the spatiotemporal profiling ofprotein signaling pathway using a microfluidic platform, combining miRNAflow-FISH with cell signaling pathway profiling will provide acomprehensive look into the relationship between miRNAs and the cellsignaling pathways they modulate.

Example 3: Protocol for miRNA Detection at Single-Cell Resolution UsingMicrofluidic LNA Flow-FISH

Flow cytometry in combination with fluorescent in situ hybridization(flow-FISH) is a powerful technique that can be utilized to rapidlydetect nucleic acids at single-cell resolution without the need forhomogenization or nucleic acid extraction. Here, we include instructionson how to set up a microfluidic device sample preparation station toprepare cells for imaging and analysis on a commercial flow cytometer ora customized built micro-flow cytometer.

Flow-FISH is a method that was first developed by adapting Q-FISH orquantitative fluorescence in situ hybridization for use in suspendedcells followed by analysis with flow cytometry to measure the length oftelomeres (Rufer N et al., “Telomere length dynamics in human lymphocytesubpopulations measured by flow cytometry,” Nat. Biotechnol. 1998;16(8): 743-7). Flow-FISH is an enormously useful technique because itprovides single-cell resolution data from a mixed population of cells,but it is labor intensive and cumbersome, requiring hybridization timesof up to several days, which makes reliable detection of vulnerable RNAspecies very challenging.

To improve the workflow as well as reliability of flow-FISH, a new classof oligonucleotide analogues called locked nucleic acids (LNA) with theribose ring constrained by a methylene bridge between the 2′-oxygen andthe 4′-carbon, has been incorporated into FISH probes to providedrastically improved hybridization characteristics (Silahtaroglu A N etal., “FISHing with locked nucleic acids (LNA): evaluation of differentLNA/DNA mixmers,” Mol. Cell. Probes 2003; 17(4):165-9). The methylenebridge in LNA molecules reduces the conformational flexibility of theribose ring, resulting in +1 and +2° C. of thermal stability per LNAmonomer in LNA/DNA mixed nucleotide probes during Watson-Crickbase-pairing (Kumar R et al., “The first analogues of LNA (lockednucleic acids): phosphorothioate-LNA and 2′-thio-LNA,” Bioorg. Med.Chem. Lett. 1998; 8(16):2219-22). The LNA containing probe's highaffinity enables the experimenter to decrease hybridization time fromdays to <2 hours, and significantly increase hybridization temperatureto reduce signal to noise ratio.

Since the advent of LNA probes, LNA flow-FISH has been adapted to detectmessenger RNA (mRNA), viral RNA, and bacterial RNA (see, e.g., RobertsonK L et al., “LNA flow-FISH: a flow cytometry-fluorescence in situhybridization method to detect messenger RNA using locked nucleic acidprobes,” Anal. Biochem. 2009; 390(2):109-14; Robertson K L et al.,“Monitoring viral RNA in infected cells with LNA flow-FISH,” RNA 2010;16(8): 1679-85; and Robertson K L et al., “Locked nucleic acid and flowcytometry-fluorescence in situ hybridization for the detection ofbacterial small noncoding RNAs,” Appl. Environ. Microbiol. 2012;78(1):14-20).

While LNA probes provide undeniable improvement to the flow-FISH method,there are other improvements to be made. The necessity of centrifugationto wash suspended cells greatly contributes to sample loss, thus makingthe detection of mammalian small non-coding RNA or other very rare RNAsdifficult, due to the need to incorporate a signal amplification stepwith many reagent changes and washes, and thus many more centrifugationsteps.

Another weakness of the existing LNA flow-FISH method is that it is notoptimized for multiplexed detection of nucleic acids along withproteins, which limits the scope of information that can be gatheredusing the technique. Until recently, only conventional FISH usingtyramid signal amplification (TSA) to amplify the small RNA signal inadherent cells or tissue sections can multiplex the detection of smallnon-coding RNAs and proteins in single cells (de Planell-Saguer M etal., Nat. Protoc. 2010; 5:1061-73).

Conventional FISH is labor intensive and time consuming, both in termsof sample preparation and analysis via microscopy. In addition, TSAamplification of miRNA signals can be difficult to reproduce due tonon-specific enzymatic amplification of signal. Here, we present amethod to significantly advance LNA flow-FISH by combining FISH withrolling circle amplification in a microfluidic sample preparation chipto integrate novel biochemistries to monitor miRNAs and proteins atsingle-cell resolution. Microfluidic sample preparation allows theelimination of centrifugation steps that contributes to sample loss, andthe minute reagent and cell requirement makes optimization of molecularassay chemistries possible, while keeping the costs to ˜5% of benchscale reactions. By using isothermal rolling circle amplification ofmiRNA signals, the microfluidic LNA flow-FISH method is fully compatiblefor multiplexing nucleic acid detection with protein immunostaining inthe same cell.

Microfluidic sample preparation have been used to study proteins,genomes, bacteria, and rare cancer cells at single-cell resolution (see,e.g., Wu M et al., Curr. Opin. Biotechnol. 2012 February; 23(1):83-8; WuM et al., Lab Chip 2012 Aug. 21; 12(16):2823-31; Liu Y et al.,“Single-cell measurements of IgE-mediated FcϵRI signaling using anintegrated microfluidic platform,” PLoS One 2013; 8(3):e60159; Yilmaz Set al., “Curr. Opin. Biotechnol. 2012; 23:437-43; Liu P et al., Lab Chip2011; 11:2673-9; and Powell A A et al., “PLoS One 2012; 7:e33788). Thechallenge to incorporate microfluidics into traditional molecularbiology lab can now be overcome with the use of commercially availablemicrofluidic chips and fluid control components.

This example provides a schematic for building and using simplemicrofluidic systems for cell analysis, with minimal need for previousexpertise in microfluidic engineering. To demonstrate the microfluidictechnique, we demonstrate the detection of miR155 and CD69 in activatedJurkat cells as illustration of the microfluidic miRNA flow-FISH method.

Reagents for On-Chip LNA FISH Sample Preparation

Unless otherwise stated, prepare all reagents in nuclease-free tubes;and use nuclease-free ultrapure water that is not DEPC treated. If it isnot possible to work in an RNase-free area, diligently wipe down allsurfaces and pipets intended for the experiment with RNase Away(Molecular Bioproducts, San Diego, Calif.) before starting theexperiment. Always wear fresh gloves and change gloves frequently toprevent RNase contamination. The volumes presented here are intended formicrofluidic chip based sample preparation.

Reagents included as follows:

0.1×SSC: 250 μl of 20×SSC with 49.75 ml of H₂O in a 50 ml conical tube,mixed well by inverting, and stored at room temperature (RT);

3 M NaCl: 8.766 g of NaCl with 25 ml of H₂O, vortexed well to dissolveNaCl completely, brought to a total volume of 50 ml with H₂O, and storedat RT;

50% formamide in 2×SSC: 1 ml of 20×SSC with 4 ml of H₂O and 5 ml offormamide in a 15 ml conical tube, and stored at 4° C. in a tube wrappedin foil;

1 M NaPi (pH 7.0): 2.68 g of Na₂HPO₄.7H₂O [=sodium phosphate, dibasic,heptahydrate] with 200 μl of H₃PO₄ [=o-phosphoric acid, 85%], brought toa 50 ml total volume with H₂O, sterile filtered, and stored at RT;

hybridization solution (makes 10 ml): 5 ml of formamide with 3.5 ml ofH₂O, 1 ml of 20×SSC, 50 μl of 10 μg/μl salmon sperm, 10 μl of 500 μg/mlyeast tRNA, and 50 mM NaPi (500 μl of 1 M stock), which was divided into500 μl aliquots, and stored at −20° C.;

10% bleach: 5 ml of bleach with 45 ml of H₂O, sterile filtered using a0.22 μm Steriflip filter (Millipore, Billerica, Mass.), and stored atRT;

50 ml of deionized H₂O, sterile filtered using Steriflip filter, andstored at RT;

1 mg/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, Mo.): 1ml of DMSO with 1 mg size vial of PMA, mixed well by vortexing afterreplacing cap, and stored as 50 μl aliquots at −20° C.;

8% paraformaldehyde (PFA): dilute 5 ml of 16% PFA (Electron MicroscopyScience, Hatfield, Pa.) with 5 ml of PBS immediately before use, where,once opened, the 16% PFA solution should be stored at 4° C. in a 15 mlconical tube wrapped in foil and be used for up to 1 week after opening;

0.1 M NaHCO₃ solution (pH 8.0): 4.1 g of NaHCO₃ with 50 ml of H₂O,sterile filtered, and stored at RT;

1 N NaOH: 4 g of NaOH with 1 L of H₂O (H₂O need not be nuclease free),stored at RT;

Solution 1 (makes 3.2 ml): 32 μl of 1-methylimidazole (stored in adesiccator) with 2.839 ml of H₂O, 320 μl of 3 M NaCl, and 9 μl of 12 MHCl, which was mixed well by vortexing and made fresh for eachexperiment;

Solution 2 (makes 1 ml): 30 mg of EDC (˜30 mg weighed out into 1.7 mleppendorf tubes and stored in a desiccator at −20° C., which was usedwithin 3 months after storage) with 1 ml of Solution 1, which was madefresh for each experiment;

Working Cell-Tak™ solution (BD Biosciences, Bedford, Mass.): 20 μl ofCell Tak™ with 570 μl NaHCO₃ (pH 8.0), which was made fresh for eachexperiment; and

500×PMA and ionomycin (Sigma-Aldrich, St. Louis, Mo.) working solution:diluted 1 mg/ml PMA in DMSO at 1:100 into PBS to make 10 μg/ml PMA,combined 50 μl of 10 μg/ml PMA with 50 μl of 1 mM ionomycin to make 500×working solution, which was made fresh each experiment.

Reagents for LNA FISH Probe

Pre-designed LNA containing ISH probes for miRNA targets can bepurchased from Exiqon A/S. When selecting probes for miRNA LNAflow-FISH, purchased LNA probes were labeled with DIG on both N and Ctermini for the strongest signal amplification. When analyzing moreabundant RNA species such as mRNAs of structural genes, only one haptenlabel per probe is sufficient. Exiqon supplied LNA probes as 25 μM stockin water. To these initial stock solutions, nuclease free water wasadded to the tube to make 10 μM LNA probe stock, divided into 5 μlaliquots, and stored at −20° C. The LNA probes for miR155 and itsscrambled control included the double DIG-labeled miR155 sequence of5/DigN/ACCCTATCACGATTAGCATTAA/3Dig_N (SEQ ID NO:1); and the doubleDIG-labeled scrambled control sequence of5/DigN/GTGTAACACGTCTATACGCCCA/3Dig_N (SEQ ID NO:2).

Antibodies Suitable for Multiplexing with LNA Flow-FISH

Quantum dot pre-labeled secondary antibodies can be purchased fromInvitrogen™ of Life Technologies, Corp. Custom labeling of primaryantibodies can be done by using one of several Q-dot antibody labelingkits, also available through Invitrogen™. Antibodies includedbiotin-labeled anti-CD69 antibody (eBiosciences, San Diego, Calif.) andQdot® 705-conjugated streptavidin (Invitrogen, Carlsbad, Calif.).

Reagents for Rolling Circle Amplification (RCA)

The RCA reaction was conducted using an antibody to bind the LNA probe,as well as additional nucleic acid sequences to form the circulartemplate and fluorophore labels for detection. In particular, reagentsfor RCA included anti-DIG antibody (Roche Diagnostics, Indianapolis,Ind.); FITC-labeled Duolink® mouse PLUS and MINUS probes (OlinkBioscience AB, Uppsala, Sweden), such as Duolink® In Situ PLA® ProbeAnti-Mouse PLUS and Duolink® In Situ PLA® Probe Anti-Mouse MINUS(Sigma-Aldrich); Green detection kit (Olink Biosciences, Uppsala,Sweden), such as Duolink® In Situ Detection Reagents Green(Sigma-Aldrich), and TBST buffer, which included 1.21 g of Tris base,4.38 g of NaCl, 0.25 ml of Tween 20, with 40 ml of H₂O. For the TBSTbuffer, the pH was adjust to 7.6, and then the total volume was broughtto 50 ml with H₂O.

Reagents for Cell Detachment

As described herein, test samples containing cells were captured byusing Cell-Tak™, a commercially available tissue adhesive containingpolyphenolic proteins extracted from Mytilus edulis (a marine mussel),which is then formulated in 5% acetic acid. These proteins can containlarge amounts of hydroxyproline (Hyp) and 3,4-dihydroxyphenylalanine(Dopa). Any enzyme or chemical agent can be employed that preferentiallycleaves these amino acids, such as Arg-C proteinase, Asp-Nendopeptidase, Asp-N endopeptidase, chymotrypsin, clostripain,collagenase, elastase (type I or type II), formic acid, glutamylendopeptidase, pepsin, proteinase K, staphylococcal peptidase I,thermolysin, and/or trypsin.

Here, reagents included elastase (Worthington Biochemical Corp.,Lakewood, N.J.); phosphate buffered solution (PBS) with Ca²⁺ and Mg²⁺(Hyclone, Logan, Utah); PBS without Ca²⁺ and Mg²⁺; and 100 mM EDTAsolution: 1 ml of 0.5 M EDTA with 4 ml of PBS without Ca²⁺ and Mg²⁺.

Components for a Microfluidic Platform

Performing microfluidic sample preparation is now possible intraditional molecular biology laboratories with little prior engineeringexpertise. Commercially available ready-to-use microfluidic chips,fittings, valves, and pumps can be purchased and configured formicrofluidic cell preparation and signal acquisition. The followingmaterials are all commercially available, and customization can beachieved through the vendors.

Microfluidic chip: A custom fused silica microfluidic chip with fourteenports, of which only thirteen ports are labeled in FIG. 3A. The designwas made using AutoCAD (Autodesk Inc. San Rafael, Calif.), photomaskswere generated at PhotoSciences (Torrence, Calif.), and fused silicachips were fabricated by Caliper Life Sciences (Hopkinton, Mass.). Othervendors may have become available for custom chip fabrications, but wehave not compared quality of chips between different vendors. The vendorwill typically aid in the formatting of the design file for custom chipfabrication. We recommend, in some situations, using fused silica orglass microfluidic chips. While other materials such as PDMS can also beused to fabricate chips, our method has been tested on glass chips.

The cell holding chamber had channel dimensions of a width of 200 μm,depth of 30 μm, and total length of about 12 cm. The dimensions ofchannels connected to sheath ports (sheath channels) included a width of30 μm and a depth of 30 μm. An array of fourteen holes, each 500 μm indiameter, seven on each side of the device, provided fluidinlets/outlets to the cell holding chambers and the sheath channels. Thecenter of the chip is shown magnified in FIG. 3A (right), where cellscan be detached after flow-FISH and hydrodynamically focused for on-chipmicro-flow cytometry. The fused silica chips can be reused after propercleaning protocol between each experiment.

Pumping system, manifold, and controller components: The microfluidicplatform also included a custom plastic chip manifold formed from anacetal resin (Delrin®); 1/32 inch o.d., 125 μm i.d. PEEK tubing(Upchurch Scientific, Oak Harbor, Wash.) for fluidic connections;fittings for PEEK tubing (Upchurch Scientific, Oak Harbor, Wash.) forfluidic connectors; fourteen custom built shut-off electronic valves,one for each port on the chip (commercial version available fromLabSmith, Livermore, Calif.); an airtight pressurizable reagentreservoir; electronic pressure controllers (Parker Hannifin, Cleveland,Ohio); a house nitrogen source; a thermoelectric hot plate (TETechnology Inc., Traverse City, Mich.); and a proportional integralcontroller for hot plate.

Micro-flow cytometer components: The micro-flow cytometer included thefollowing optical components to deliver an excitation source and measureresultant signals from the probes. The components includes a 20-mW diodepumped solid state laser at 488-nm (85-BCF-020-112; CVI Melles Griot,Carlsbad, Calif.) in an epifluorescence configuration for excitation; along pass dichroic mirror (LPD01-488S; Semrock, Rochester, N.Y.); anaspheric lens (5722-H-B; New Focus, Santa Clara, Calif.); aphotomultiplier tube (PMT)-based detector (H5784-20; Hamamatsu,Bridgewater, N.J.); a custom-made sculpted tip silica optical fiber(1000 μm core, 2000 μm sculpted spherical tip; Polymicro Technologies,Phoenix, Ariz.); an eight channel Hamamatsu linear multi-anode (LMA) PMTcoupled with filter optics (H9797TM; Hamamatsu, Bridgewater, N.J.);three dichroic mirrors (DMT560, DMT650, and DMT740, Hamamatsu,Bridgewater, N.J.); and four bandpass filters (BPF534_30, BPF585_40, andBPF692_40, and BPF785_62, Semrock, Rochester, N.Y.).

Methods for Microfluidic Chip Setup, Cleaning, and Surface Modificationfor Reversible Cell Capture

To prime the chip, sterile filtered deionized water was pre-loaded intoeach channel by placing a water droplet of ˜100 μl onto an inlet portand allowing capillary forces to drive water into the microfluidicchannel. Vacuum can be applied to an opposite port to facilitate watertransport into the microchannel, thereby filling the microchannels withas much water as possible before assembly of the chip into the manifold.

FIG. 3A shows a device with various ports, and FIG. 3B shows asimplified schematic for microfluidic sample preparation station 3000.We employed custom-fabricated microvalves, reagent reservoirs, chipmanifold, and computer control unit. Of course, commercial versions ofthe microvalves, reservoirs, and chip connectors can be purchased from,e.g., Fluigent SA (Paris, France) or LabSmith, Inc. (Livermore, Calif.).Some initial customization may be required, and the vendors should aidin the design and optimization of that initial customization. We havebuilt a fourteen-valve setup with temperature control to accommodate theten chamber microfluidic device 300 (shown in FIG. 3A, left). Eachchamber 321-330 can hold up to 2000 cells and are fluidically isolatableto enable ten different experimental conditions in one chip experiment.An array of ports 310-320, 351, 360 serves as inlets and outlets forfluid connections to the chip's microchannels. The center of the device(FIG. 3A, right) provides the region in which cells can behydrodynamically focused into single file for on-chip laser inducedfluorescence and flow cytometry. The number of chip ports, valves,reservoirs, and pressure controllers can be customized for individualend user groups.

To set up the device (chip) 3010 (FIG. 3B), the custom manifold wasvisually aligned to the chip, and each port was connected to one end ofthe PEEK tubing via Upchurch fittings. The electronic shut-off valves3061-3064 were used in-line with the tubing to control the movement offluid into each port. The other end of the PEEK tubing was submerged inpreloaded, airtight reservoirs M31-M34 and pressurized using housenitrogen and electronic pressure controllers 3031-3034. The pressurecontrollers 3031-3034 generate regulated pressure (arrows in inset ofFIG. 3B) that is delivered into the reservoirs M31-M34.

By applying air into the sample reservoir M31 (FIG. 3B, inset), thepositive pressure generated by the air drives fluid out through thelonger tubing that is submerged in the preloaded reagent/sample and tothe in-line valve 3061. In this manner, fluid can be moved, in acontrollable manner, through tubing and reach programmable valves3061-3064 that are connected in-line between the reservoirs M31-M34 andthe microfluidic device 3010. The velocity at which the fluid flow intothe chip is controlled by the pressure controllers 3031-3034. A greaterpressure difference generates a faster fluid flow velocity and,therefore, higher shear stress inside the microfluidic channel.

The valves 3061-3064 and pressure controllers 3031-3034 can becontrolled by a master controller (an integration hub 3040), which inturn is operated by way of a processor 3080. The user can control whichport on the device the fluid can access by controlling the opening andclosing of the in-line valves 3061-3064. For instance, an integrationhub 3040 can connect the programmable valves and pressure controllers toa central computer 3080, where the user can script operational programsto automate sample preparation sequences, such as washing and stainingof cells in the microfluidic device. A heat plate with temperaturecontrol can be added to facilitate incubation at higher temperatures.

Once assembled, the device can be prepared by filling with water. First,about 1 ml of water was introduced into the main port 310 (in FIG. 3A),which allows each chamber 321-330, flow cytometry channel 355, andsheath channel 356-357 to be filled with water, thereby reducing airbubbles within the channels. To load samples and/or reagents into thedevice, positive pressure from house nitrogen was used to transport thefluid from the reservoir and to the port, which in turn was in fluidiccommunication with the chambers and channels of the device. Pressure canbe adjusted using the pressure controllers and, generally, pressuresbetween 0.2 psi to 15 psi were used for the experiments describedherein.

To address a specific cell chamber, various ports were opened and closedto provide a fluidic pathway (for the reagent) that led to the desiredcell chamber. For instance, the main port 310 can be used to loadreagents into the device, and valves can be controlled to provide thedesired fluidic pathway. This can be achieved by opening the valve influidic communication to port 310, opening the valve to a port influidic communication with the targeted chamber and providing the lowestpressure difference between the main port 310 and the targeted chamber,and then closing all other valves. Then, to introduce the reagent intothe device, the pressure controller is used to apply house nitrogen to apressurized reservoir connected to port 310, and the reagent in thereservoir will move from the reservoir into the target chamber. Forexample, to the target chamber 321, one can first shut off all valves,apply pressure to the main port 310, and open the valves to the mainport 310 and the target port (i.e., the port 311, which when opened willprovide the lowest pressure difference between the main port 310 and thetarget chamber 321), thereby ensuring that the preferential fluidicpathway will require transport of the reagent from the main port 310,through the target chamber 321, and exiting out of the target port 311.

The device can be cleaned for reuse. For instance, the device can becleaned by flushing all channels with a 10% sterile filtered bleach indeionized water for 10 min. at 10 psi at RT, followed by a 10 min. flushwith deionized water for 10 min.

For cell capture, the working Cell-Tak™ solution can be employed. First,10 μl of 1 N NaOH was added to the working Cell-Tak™ solution within 10min. of introducing the solution into the device. If the pH of thecoating buffer is not between 6.5-8.0, Cell-Tak™ will not performoptimally. An aid to attaining this pH window is to use a volume of 1 NNaOH equal to half the volume Cell-Tak™ solution, used in combinationwith a neutral buffer. For example, one methodology includes 10 μl ofCell-Tak™, 285 μl of sodium bicarbonate (pH 8.0), and 5 μl of 1 N NaOH(added immediately before coating) to make 300 μl of the workingCell-Tak™ solution. About 1 ml of the working Cell-Tak™ solution isadded into the reagent reservoir corresponding to the main port 310. Allthe valves are then opened to facilitate and flow of the workingCell-Tak™ solution from the main port 310 to all other ports at 15 psifor 5 min., and then pressure is reduced to 1.5 psi to maintain flow for20 min.

Next, the reservoir tube with Cell-Tak™ solution was replaced with 1 mlof sterile filtered PBS. PBS was transported through all channels for 10min. at 10 psi to flush out residual Cell-Tak™ solution. Flow wasstepped, and the coated device should be used within one day.

Methods for Constructing the Micro-Flow Cytometer

While setting up the device and fluid control station can be done withminimal engineering knowledge, building a custom micro-flow cytometerrequires high technical expertise in mechanical and optical engineering.If no such engineering capabilities are available, samples can bedetached from the chip and analyzed using a commercial flow cytometer.If using conventional flow cytometer for analysis, this followingconstruction method can be omitted.

A simplified schematic of the micro-flow cytometer 3100 is shown in FIG.3C. The optical fiber 3111 and PMTs 3171-3174 are aligned vertically tothe area on the device 3110 where cells are hydrodynamically focusedinto a single file line. As the cells pass through the laser beam, thePMTs record scatter and laser induced fluorescence signals coming off ofthe cells.

To build the micro-flow cytometer, we employed a 20-mW diode pumpedsolid state laser at 488-nm 3160 in an epi-fluorescence configurationfor excitation. The laser 3160 was placed beneath the device 3110, andthe laser beam was aligned to reflect and focus upon the detectionregion at the center of the device 3110 by using a long pass dichroicmirror and an aspheric lens. An optical fiber 3111 with a PMT 3120 wasinstalled and aligned on top of the device 3110, where the alignmentposition should be slightly behind the path of the laser beam to collectforward scatter from passing cells.

Beneath the device 3110, an optical system was installed to provide anexcitation source, as well as to configure the detector. First, anaspheric lens 3130, dichroic mirror, and PMT modules 3171-3174 wereinstalled beneath the device 3110 so that scatter and fluorescencesignals from passing cells will first be focused by the lens 3130 andsubsequently passed via the dichroic mirror for detection in the PMTmodule.

For multiplexed fluorescence detection, we employed an eight channelHamamatsu linear multi-anode (LMA) PMT coupled with filter optics. For afour color detection setup, we used four channels of the LMA PMT andselected the filtering for green, yellow, red, and far-red fluorescencedetection. Bandpass filters 3175 were used for selected wavelengths.

For green fluorescence detection, we cascaded a portion of the aggregateflorescence signal upon the first LMA PMT channel via a longpassdichroic mirror and filtered through to the detection region of thatchannel using a bandpass filter (BPF534_30). For yellow fluorescencedetection, we cascaded a portion of the remaining florescence signalupon the second LMA PMT channel using another longpass dichroic mirror(DMT650) and filtered through to the respective channel's detectionregion using a second bandpass filter (BPF585_40). For red fluorescencedetection, we used the third channel of the LMA PMT via a third longpassdichroic (DMT740) and filtered using a third bandpass filter(BPF692_40). For far-red fluorescence detection, we cascaded theremaining florescence signal onto the fourth LMA PMT channel using amirror, and filtered onto that channels detection region using a fourthbandpass filter (BPF785_62).

Methods for miR155 Detection in PMA and Ionomycin Activated Jurkat Cells

Jurkat cells (ATCC, Manassas, Va.) were cultured in RPMI supplemented by10% FBS (Hyclone, Logan, Utah) in a humidified cell culture incubator,which was maintained at 5% CO₂ and 37° C. Then, cells were seeded at10⁶/ml at 1 ml per well into a 24 well plate. Stimulation experimentswere conducted by incubating Jurkat cells for 0, 8, 16, 20, or 24 hourswith 10 ng/ml PMA and 1 μM ionomycin. About 2 μl of 500×PMA andionomycin working solution was added to each well and then swirled tomix. Incubation for designated lengths of time was conducted in the cellculture incubator.

At designated times, Jurkat cells were transferred to Eppendorf tubes,centrifuged at 300 g for 5 min., and resuspended with 1 ml of 8% PFAsolution for each tube, and incubated at RT for 10 min. Fixed cells werepelleted at 300 g for 5 min., resuspended with 1 ml of PBS for eachtube, and incubated at RT for 5 min. This last step was then repeated.Then, the washed cells were pelleted at 300 g for 5 min. and resuspendedin 50 μl of PBS to make 2×10⁷ cells/ml for loading onto the chip.

Jurkat cells were loaded into reservoirs and transported within aCell-Tak™ surface modified device at 5 psi for 3 min. The followingports were used to provide the desired fluidic pathway that resulted incapture of cells in the desired cell chamber: for 0 h. cells—from port311 to exit port 360, port 320 (to fill chambers 321, 330); for 8 h.cells—from port 312 to exit port 360, port 319 (to fill chambers 322,329); for 16 h. cells—from port 313 to exit port 360, port 318 (to fillchambers 323, 328); for 20 h. cells—from port 314 to exit port 360, port317 (to fill chambers 324, 327); and for 24 h. cells—from port 315 toexit port 360, port 316 (to fill chambers 325, 326). After targeting thedesired cell chambers, flow was stopped, and the cells were incubatedfor 15 min. at RT to allow capture onto the surface of themicrochannels.

Methods of CD69 Cell Surface Protein Immunostaining

On-chip cell surface protein immunostaining was performed as follows.Immediately after cell loading, a 1:50 dilution of anti-CD69-biotinantibody (1 μl antibody with 49 μl of TBS) was prepared. Then, theantibody solution (a protein label) was transported through the mainport 310 and into each chamber at 5 psi. After 2 min., flow was stopped.The antibody solution was incubated for 30 min. at RT. Next, a TBSTbuffer was transported through the channels at 2 psi for 5 min. Theprobe having a detectable marker (Qdot® 705-labeled streptavidin) wasdiluted at 1:50 in TBS (1 μl Qdot®-705 streptavidin in 49 μl TBS). Thissolution was then transported to the channels at 5 psi for 2 min., flowwas then stopped, and the device was incubated at RT for 30 min.Finally, the probe was rinsed through the device by flow of TBST throughthe channels at 2 psi for 5 min.

Methods for In Situ Hybridization with miR155 and Scrambled LNA Probes

On-chip cell miRNA detection was performed as follows. Captured,immunostained cells were permeabilized by flowing 0.25% Triton™ X-100 inTBS to each chamber. Flow was stopped, and the device was incubated for10 min. at RT. Cells were washed with TBS for 10 min. at 3 psi. and thenwith Solution 1 (described herein) for 5 min. at 5 psi. Cells were thenincubated with Solution 2 (described herein) for 30 min. at RT, and thenwashed with TBS for 5 min. at 5 psi. Pre-warmed hybridization solution(to 65° C.) was transported into all chambers for 5 min. at 5 psi,followed by incubation for 1 h. at 62° C. for pre-hybridization todecrease non-specific hybridization.

During the 1 h. pre-hybridization phase, 2 μM LNA probe solutions inhybridization buffer (5 μl of 10 μM LNA stock with 20 μl ofhybridization buffer) were prepared. After the 1 h. pre-hybridizationphase, the prepared LNA probe solutions were transported into chambersfor 3 min. at 5 psi.

The following ports were used to provide the desired fluidic pathwaythat resulted in labeling with the miR155 probe in the desired cellchamber: from port 311 to port 312 (to fill chambers 321, 322), port 313(to fill chamber 323), port 314 (to fill chamber 324), port 315 (to fillchamber 325), and exit port 360 (to prevent overflow into chambers326-330). The following ports were used to provide the desired fluidicpathway that resulted in labeling with the scrambled probe in thedesired cell chamber: from port 320 to port 319 (to fill chambers 330,329), port 318 (to fill chamber 328), port 317 (to fill chamber 327),port 316 (to fill chamber 326), and exit port 360 (to prevent overflowinto chambers 321-325). After establishing the desired fluidic pathway,flow was stopped to allow for hybridization at 80° C. for 90 s.,followed by incubation at 62° C. for 90 min. Then, the chambers werewashed with 2×SSC with 50% formamide at 65° C. for 10 min., where flowwas established for 5 min. at 5 psi and then stopped to allow forincubation for 5 min.). Successive washes included washing with 1×SSCfor 20 min. at RT (flow for 5 min. at 5 psi, stop flow, and incubate for5 min.) and with 0.1×SSC for 20 min. at RT (flow for 5 min. at 5 psi,stop flow, and incubate for 5 min.).

Methods for miR155 Signal Amplification Using Proximity Ligation andRolling Circle Amplification

All reagents for RCA amplification, with the exception of the anti-DIGantibody, were part of the Duolink® mouse PLUS kit, mouse MINUS kit, andgreen detection kit from Olink AB (Uppsala, Sweden). The kits included aDuolink® Blocking solution (1×) (including5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazolin-3-one(3:1 mixture) and bovine serum albumin); a Duolink® Diluent solution(1×) (including5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazolin-3-one(3:1 mixture) and bovine serum albumin); Duolink® PLA probe anti-mouseMINUS/PLUS (including the probe,5-chloro-2-methyl-4-isothiazolin-3-one/2-methyl-4-isothiazolin-3-one(3:1 mixture), and bovine serum albumin); Duolink® Ligase (1 U/μl)(including dithiothreitol (DTT)); Duolink® Ligation solution (5×)(including tris hydrochloride, tris(hydroxymethyl)aminomethane, and DTT,as well as all components needed for ligation (e.g., oligonucleotidesthat hybridize to the PLA probes, such as connector probes) except theligase); Duolink® Polymerase (10 U/μl) (including DTT); Duolink®Amplification solution (5×) (including tris(hydroxymethyl)aminomethane,as well as all components needed for RCA (e.g., oligonucleotide probeslabeled with a fluorophore that hybridize to the RCA product) except thepolymerase).

Enzymes were transported on ice during the experiment to preventdegradation. When the anti-DIG antibody was first used, the antibodysolution was divided into working 5 μl aliquots and stored at −20° C. toavoid repeated freeze/thaw cycles. All unconjugated antibodies should bestored as working aliquots at −20° C.

The RCA assay was performed on-chip as follows. First, the Duolink®Blocking solution was transported to each chamber in the device for 5min. at 5 psi. Flow was then stopped to allow incubation for 30 min. at37° C. Then, the anti-DIG antibody solution (diluted at 1:50 with 1 μlof anti-DIG antibody and 49 μl of PBS) was transported into all chambersand incubated for 30 min. at RT. The chambers were then washed with TBSTfor 5 min. at 5 psi at RT. Next, Duolink® PLA probe anti-mouse MINUS andDuolink® PLA probe anti-mouse PLUS agents (diluted at 1:5 with 10 μl ofPLUS, 10 μl of MINUS, and 30 μl of Duolink® Diluent solution), weretransported into all chambers for 3 min. at 5 psi. Flow was stopped toallow incubation for 30 min. at 37° C. Again, chambers were then washedwith TBST for 5 min. at 5 psi at 37° C.

Ligase and polymerase reactions were then conducted to form the circulartemplate and to form the concatemer based on this template. First, aligase solution (diluted at 1:40 with 8 μl of Duolink® Ligation solution(5×), 31 μl of water, and 1 μl of Duolink® Ligase (1 U/μl)) wastransported to all chambers at 5 psi for 3 min. Flow was stopped toallow incubation for 30 min. at 37° C. Then, chambers were washed withTBST for 5 min. at 5 psi at 37° C. Next, a polymerase solution (dilutedto 1:80 with 8 μl of Duolink® Amplification solution (5×), 32 μl water,and 0.5 μl of Duolink® Polymerase (10 U/μl)) was transported to allchambers for 3 min. at 5 psi. Flow was stopped to allow incubation for 2h. at 37° C. Cells were then fixed by transporting 1% PFA in TBS to eachchamber for 5 min. at 5 psi at RT. Finally, all chambers were washedwith TBS for 5 min. at 5 psi at RT.

At this point, cells were ready for imaging. The amplified miR155 signalappeared as fluorescent green dots in the cytosol, and the Qdot®-labeledCD69 protein was visualized in the same cell. See FIG. 6A as an exampleof a multiplexed miRNA and protein detection in the same cell. Thereshould be visible incremental increase in miR155 signal, as well as CD69signal as time of stimulation with PMA and ionomycin increases.

Methods for Cell Detachment

After on-chip sample preparation and image acquisition, cells can bedetached from the Cell-Tak™ modified channel surface by a combination ofproteolytic cleavage and shear force. First, a 100 μg/ml elastasesolution was prepared in PBS containing Ca²⁺ and Mg²⁺. Then, thiselastase solution was transported into each chamber and incubated for 5min. at 42° C., followed by flowing elastase solution for 5 min. at 15psi into collection tubes containing 100 μl of 100 mM EDTA in Ca²⁺/Mg²⁺free PBS (20 μl of 0.5 M EDTA and 80 μl of PBS).

For each cell holding chamber, detached cells were transported towardsexit port 360. At the same time, PBS was transported from sheath port351 to exit port 360 to act as sheath fluid to hydrodynamically focusthe detached cells from the holding chambers for on-chip flow cytometry.Samples can be analyzed by using the micro-flow cytometer, as describedherein, or a commercial flow cytometer, as also described herein.

Methods for Cell Analysis Using Micro-Flow Cytometer

The micro-flow cytometry was performed at the center of the chip (FIG.3A, right), where the sheath fluid focuses the sample stream containingthe detached cells into the path of the laser beam coming fromunderneath the chip. To hydrodynamically focus the sample portion, thecells were first detached by transporting the elastase solution from aport corresponding to the desired chamber to the exit port 360 at 15psi. For instance, to analyze a sample portion from chamber 321, theelastase solution can be transported from port 311 to exit port 360.

Then, sheath fluids were transported to the flow cytometry channel byflowing a sheath fluid (e.g., PBS) from sheath port 351 to exit port 360at 5 psi to hydrodynamically focus the cells. The pressure at the sheathport 351 can be adjusted between each sample to ensure that focusing isconsistent across all chambers. The pressure settings for creatingconsistent hydrodynamic focusing between chambers can be determinedempirically by using 10% glycerol solution in PBS in the sheath channel.For instance, the glycerol solution can be transported into sheath port351 and then towards exit port 360 at 5 psi. Then, PBS can betransported through each assay chamber one at a time and towards exitport 360 at 15 psi. Flow characteristics can be determined visually toassess much pressure to apply at sheath port 351 so that the pinched PBSstream remains in the center of the channel and at a consistent width.

Detectable signals can be collected using the optical system, as shownin FIGS. 2D and 3C. For instance, laser-induced forward scatter andfluorescence signals can be collected from the passing cells with theoptical fiber and PMT located above the device. Multiple spectra can bedetected by collecting the scatter and fluorescence signals for allchannels (488-nm scatter, green, yellow, red and far-red) by the dataacquisition module located beneath the device. Histograms of scatter andfluorescence data were generated with each cell passing through thedetection region (dashed circle in FIG. 3A, right), and peaks from eachhistogram were identified (e.g., using LabVIEW to script a “peak finder”application) from the scatter and fluorescence histograms to generate anumerical value representing each cell. Peak values were then exported(e.g., into either Excel or Kaleidagraph) and plotted in each sample setto generate histograms for each experimental conditions. Generatedhistograms were overlaid to show changes in the fluorescence betweeneach experimental condition.

Methods for Flow Cytometric Analysis Using Commercial Flow Cytometer

Instead of an on-chip micro-flow cytometer, analysis can be conductedoff-chip by using a commercial flow cytometer. This method includespre-labeling new collection tubes with sample identification informationand aliquoting 100 μl of 100 mM EDTA solution into each collection tube.The tubes should be stored on ice for at least 10 min. prior to celldetachment. On-chip cells can be detached (e.g., as described herein)and collected from each chamber in its own collection tube. Detachedcells should be immediately stored on ice. Within each collection tube,the total volume can be brought up to 200 μl with an ice cold 100 mMEDTA solution and then immediately analyze in commercial flow cytometer.

Example 4: Microfluidic Molecular Assay Platform for the Detection ofmiRNAs, mRNAs, Proteins, and Post-Translational Modifications atSingle-Cell Resolution

Since we know that cells in populations behave heterogeneously (see,e.g., Wu M et al., Curr. Opin. Biotechnol. 2012 February; 23(1):83-8),especially in the cases of stem cells, cancer, and hematopoiesis, thereis need for a new technology that provides capability to detect andquantify multiple categories of signaling molecules in intact singlecells to provide a comprehensive view of the cell's physiological state.In this example, we describe an exemplary, automated microfluidicplatform with a portfolio of customized molecular assays that can detectnucleic acids, proteins, and post-translational modifications in singleintact cells with >95% reduction in reagent requirement in under 8hours.

Cell signaling is a dynamic and complex, intricate process that involvesmany players, including proteins, nucleic acids, and transientpost-translational modifications (PTMs) (FIG. 7). The traditional viewof the cell signaling cascade begins with cell surface receptoractivation 701 of the cell 700, where a change in receptor structureconformation begins a cascade of events 702 (e.g., a kinase cascade,where kinases transiently phosphorylate in series), leading to thetranslocation of transcription factor into the nucleus to induceexpression of relevant genes into messenger RNA (mRNA) 703. The inducedmRNA is exported into the cytosol and translated into proteins 705 thatcan then carry out response to the original stimulus that activated thesurface receptor.

To make the process even more complex, miRNAs can be induced along withthe mRNA 703, and the miRNAs function by binding the 3′ untranslatedregion (UTR) and recruit the RISC complex to degrade the mRNA 704(Krutzfeldt J et al., Cell. Metab. 2006; 4:9-12). In this manner, miRNAcan modulate mRNA expression in the cytosol.

Signaling pathways involve proteins, miRNA, and mRNAs, along withvarious forms of transient post-translational modifications, anddetecting each type of signaling molecule requires categoricallydifferent sample preparation methods such as Western blotting forproteins, PCR for nucleic acids, and flow cytometry forpost-translational modifications. For instance, analysis of each classof biomolecules currently requires categorically different methods forsample preparation and detection. Proteins are primarily detected byWestern blotting, nucleic acids by PCR, and PTMs by flow cytometry ormass spectrometry. The multiple sample preparation techniques requiredto detect different categories of molecular targets involvelabor-intensive steps, including stimulation, fixation,permeabilization, immunostaining, and multiple wash steps in between.The manual methods for performing large-scale cell signaling studies notonly involve bulky equipment but also many opportunities foruser-introduced artifacts that can confound reproducibility.

The analysis of cellular signaling is further complicated by the factthat cells are heterogeneous in nature. To fully understand howindividual cells respond to stimuli in cellular and disease processes,one must study the changes in protein, mRNA, miRNA, and PTMs at thesingle-cell level. For example, to study the CD8 T-cell response todifferent vaccines requires distinguishing T-cell subtypes based on cellsurface markers to study the heterogeneous gene expression patterns inindividual T cells that underlie differential induction ofvaccine-induced immunity (Flatz L et al., “Single-cell gene-expressionprofiling reveals qualitatively distinct CD8 T cells elicited bydifferent gene-based vaccines,” Proc. Nat'l Acad. Sci. USA 2011 Apr. 5;108(14):5724-9). Bulk profiling methods only generate averaged signalingmeasurement from heterogeneous cell populations, and single-cellresolution analysis of miRNA, mRNA, and protein will yield informationotherwise unattainable using bulk methods. Existing single-cell analysismethods include amplification-based methods that include PCR-basedamplification to profile DNA and RNA, but these methods can introducebias during the amplification and require days of hands-on manipulationtime to accomplish. In addition, amplification-based single-cellanalysis methods require lysis and nucleic acid extraction and cannot beused practically to profile proteins and PTMs along with the nucleicacids in the same cell.

Understanding the role of all these key biomolecular players insignaling networks on a single-cell level is critical for decipheringthe molecular mechanisms of cellular and disease processes. Such a studyrequires an integrated, multiplexed platform that enables asystems-level investigation of the complex interactions between thesesignaling biomolecules. To date, no technology exists that can integratethe detection and analysis of all the signaling molecules, particularlyat the single-cell level.

To address the need for a new technology that provides true single-cellresolution multiplexed signaling analysis, we have developed anautomated, microfluidic platform (FIG. 2A-2D) with accompanyingmolecular assays that enable rapid processing of intact cells (˜8 h) tosimultaneously detect miRNAs, mRNAs, proteins, and PTMs at single-cellresolution, with only ˜270 nL reagent and ˜1000 cells required perchamber.

The molecular assays that accompany the platform combine in situhybridization (ISH) and immunostaining to gain access to both nucleicacid and protein targets without the need to lyse and amplify cellularsignals. An added benefit is that both ISH and immunostaining arecompatible with microscopy for visual inspection of the spatiotemporalcorrelation between the proteins and nucleic acid species, while thesame sample can be analyzed in a flow cytometer to gather quantitativedata regarding the magnitude of the cellular signals.

The entire multiplexed single-cell detection platform is designed withgraphic user interface-based software (FIG. 2C) that controls all valvesand pumps, allowing for hands-free operation of the platform from cellloading and stimulation to sample preparation, followed by detection andautomated analysis (FIG. 2D). The elimination of manual cell handlingsteps greatly reduces time and labor, and the microfluidic formatreduces sample and reagent requirement by ˜95%, with the added benefitof eliminating user error. The automated platform provides systems-levelhigh-content profiling of mRNA, miRNA, and proteins in an integratedexperimental space.

In this example, we highlight the molecular assays developed on themicrofluidic platform that allow for the automated detection of miRNA,mRNA, and proteins, as well as the work flow that allows multiplexeddetection of different categories of biomolecules in the same cell.

Materials and Methods

Materials and methods were similar to Examples 1-3, unless otherwiseindicated.

Cell culture and stimulation: The RAW 264.7 murine macrophage cell linewas purchased from ATCC (Manassas, Va.), cultured in growth mediumconsisting of 450 ml of Dulbecco's modified Eagle's medium (DMEM), 50 mlof fetal bovine serum (FBS) (100-500, Gemini, West Sacramento, Calif.),10 ml of HEPES, 5 ml of L-glutamine (200 mM), 1:100penicillin/streptomycin (Gibco, Carlsbad, Calif.), and 200 μg/mlGeneticin (InvivoGen, San Diego, Calif.). RAW cells were captured in themicrofluidic device and stimulated with 100 nM Escherichia coli smoothlipopolysaccharide (LPS) (L4524; Sigma-Aldrich, St. Louis, Mo.) ingrowth media for various times.

Microfluidic assays for cell surface protein expression, cytosolicphosphoproteins, and cytokine staining: For protein immunostaining,cells were cultured for up to 4 h. with 1 μL/ml Golgi-Plug reagentcontaining brafeldin A (555029; BD Biosciences) and fixed withparaformaldehyde (1.5%-8%) for 10 min.; incubated withfluorescent-labeled antibody targeting cell surface receptors, TLR4/MD2receptor at 1:15 dilution (117605; BioLegend, San Diego, Calif.) or CD69early T-cell activation marker at 1:100 dilution (13-0699-80;eBioscience, San Diego, Calif.) for 15 min; and washed for 5 min. withPBS. Following cell surface staining, cells were permeabilized with 0.1%Triton™ X-100 and incubated with intracellular phospho-specific ERK1/2at a 1:15 dilution (4375; Cell Signaling Technology, Danvers, Mass.) andintracellular tumor necrosis factor-α (TNF-α) at a 1:50 dilution(19-732181; eBiosciences) for 30 min.

Microfluidic mRNA and miRNA ISH: Double DIG-labeled locked nucleic acid(LNA) probes for miRNA 155, scrambled miRNA control, and an N-terminalbiotin-labeled β-actin mRNA LNA probe were purchased from (Exiqon,Vedbaek, Denmark). The following LNA-containing probes were used formiRNA and mRNA detection:

miR155: /5DigN/ACCCCTATCACGATTAGCATTAA/3Dig_N/(SEQ ID NO:1);

Scrambled: /5DigN/GTGTAACACGTCTATACGCCCA/3Dig_N/(SEQ ID NO:2);

β-actin (hsa): biotin-5′-CTCATTGTAGAAGGTGTGGTGCCA-3′ (SEQ ID NO:3); and

Scrambled: biotin-5′-GTGTAACACGTCTATACGCCCA-3′ (SEQ ID NO:4).

After performing protein immunostaining as described in the previoussection, the miRNA and mRNA ISH was performed on the same cells. Theamplification method was performed as described previously (Wu M et al.,PLoS One 2013; 8(1):e55044). Briefly, the following ISH reagents weremade fresh: Solution 1 (0.13 M 1-methylimidazole, 300 mM NaCl, pH 8.0,adjusted with HCl); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)solution (Solution 2, i.e., 0.16 M EDC in Solution 1, adjusted to pH8.0); and hybridization buffer (50% formamide, 2× saline-sodium citrate(SSC) buffer, 50 μg/ml yeast transfer RNA (tRNA), 50 μg/ml salmon sperm,and 50 mM NaPi). The immunostained cells were washed with Solution 1 for5 min, followed by incubation with Solution 2 for 20 min., and thenwashed for 5 min. with Tris-buffered saline (TBS). The cells were thenpre-hybridized for 30 min. at 62° C. in hybridization buffer pre-warmedto 65° C.

All LNA probes were used with the 10-pmol/25-μL hybridization buffer andflown into pre-determined chambers. Hybridization with LNA probes wasperformed for 90 s. at 80° C., followed by 90 min. at 62° C., which wasthen followed by the following washes: 2×SSC+50% formamide for 10 min.at 65° C., 1×SSC for 20 min. at RT, and finally 0.1×SSC for 5 min. atRT.

For the β-actin mRNA, the hybridized biotinylated LNA probe can bedetected by incubation with PE-conjugated streptavidin at 1:200 (S-866;Life Technologies, Carlsbad, Calif.) for 30 min., followed by a 5 min.TBS wash. For miRNA signal, proceed to rolling circle amplification.

Microscopy: Brightfield, epifluorescence, and phase contrast images werecaptured at ×60 and ×100 magnification on an Olympus (Tokyo, Japan)IX-71 inverted microscope equipped with a CoolSNAP HQ CCD camera(Photometrics, Tucson, Ariz.) and Image-Pro software (Media Cybernetics,Bethesda, Md.).

Results and Discussion

miRNA detection: The most technically challenging molecular target todetect in an intact single cell is miRNA (FIG. 8A). To detect miR-NAs atsingle-cell resolution, a novel flow cytometry compatible fluorescent insitu hybridization (flow-FISH) assay to detect miRNAs usingLNA-containing probes has been developed in our laboratory (Wu M et al.,PLoS One 2013; 8(1):e55044). The LNA flow-FISH assay combines theadvantage of LNA's high melting temperature (Silahtaroglu A N et al.,Mol. Cell. Probes 2003; 17(4):165-9) with the specificity of proximalligation and rolling circle amplification (Söderberg O et al., “Directobservation of individual endogenous protein complexes in situ byproximity ligation,” Nat. Methods 2006 December; 3(12):995-1000) toprovide highly specific amplification of otherwise undetectable miRNAsignals in an intact cell, which then can be visualized via microscopyand quantified using flow cytometry. The detection of miRNA species byLNA-flow FISH can be multiplexed with mRNA detection, and the isothermalnature of rolling circle amplification makes multiplexing with proteinimmunostaining possible.

mRNA detection: The LNA flow-FISH method can easily be used to detectmRNA (FIG. 8B), where β-actin mRNA was detected using biotinylatedLNA-containing probes. For abundant mRNA targets, no further enzymaticsignal amplification is required. The mRNA-probe complex can be detectedby incubating with fluorescent-labeled streptavidin. The bindingpartner-based (e.g., biotin-streptavidin-based) mRNA detection can bemultiplexed with miRNA detection.

Cell surface protein detection: To demonstrate the detection of cellsurface proteins, we used fluorescent dye-labeled antibodies directedagainst cell surface proteins after initial paraformaldehyde fixation ofthe cells but prior to permeabilization with Triton™ X-100. In FIG. 8C,the cell surface receptor complex TLR4/MD2 was activated by LPS. As theTLR4/MD2 complex changed conformation as a result of TLR4 activation,the complex lost affinity to the antibody directed against the inactiveTLR4/MD2 complex. As a result, the fluorescence associated with theinactive TLR4/MD2 complex decreased as a function of time after additionof LPS.

Protein phosphorylation profiles: FIG. 8D illustrates the detection oftransient phosphorylation in signaling proteins by demonstrating thedetection of phosphorylated p38 protein after LPS stimulation ofmacrophages. p38 is a known component of the TLR4 innate immune pathway,and the phosphorylation of p38 has been shown to lead to induction ofproinflammatory genes (Bode J G et al., “The macrophage response towardsLPS and its control through the p38(MAPK)-STAT3 axis,” Cell. Signal.2012 June; 24(6):1185-94). By using phospho-specific antibodiesconjugated to fluorescent dyes, any phospho-profiling of kinase cascadescan be performed using the platform (see, e.g., Srivastava N et al.,Anal. Chem. 2009 May 1; 81(9):3261-9).

Cytosolic protein expression profiles: Intracellular proteins can bedetected using fluorescent conjugated antibodies. To detect proteinsthat are released from the cell, such as chemokines and cytokines, aGolgi release inhibitor such as brefeldin A can be added to the culturemedia for up to 8 h. to increase the intracellular concentration ofcytokines for detection by immunostaining. In FIG. 8E, LPS-activatedmacrophages were treated with brefeldin A for 4 h., and theintracellular TNF-α protein was detected using the anti-TNF-α antibody.

Dynamic glycosylation profiles: Post-translational modifications, suchas glycosylation, provide an important biomolecular mechanism to inducevarious cellular responses. For instance, nucleoporin 62 (Nup62) is anuclear pore protein complex that mediates RNA and protein transportthrough the nuclear envelope. Both glycosylation and phosphorylation ofNup62 influences the conformation of this complex and could mediate poreassembly and disassembly. Transient and/or dynamic glycosylationprofiles can be detected on-chip, as seen in FIG. 8F.

Multiplexed assay scheme for all biomarkers: All microfluidic molecularassays developed on the platform are compatible for multiplexing witheach other and will provide systems-level analysis of signaling pathwaysin the native cellular context at single-cell resolution. Immunostainingthe protein biomarkers prior to EDC fixation and ISH of nucleic acidprobes allows for the multiplexing of protein and nucleic acid targetsin the same samples, and the entire multiplexed assay can be completedin 8 h. (FIG. 12). The use of directly conjugated antibodies allows forthe multicolor flow cytometric analysis of all biomarkers at once. Theplatform and accompanying assays will advance the knowledge of cellsignaling pathways and their correlation with disease states, and itholds great potential for both basic cell signaling research and thedevelopment of multiplexed miRNA/mRNA/protein biomarker panels fordisease diagnostics and companion diagnostics.

Other Embodiments

All publications, patents, and patent applications, including U.S.Provisional Application No. 61/918,402, filed Dec. 19, 2013, mentionedin this specification are incorporated herein by reference to the sameextent as if each independent publication or patent application wasspecifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

The invention claimed is:
 1. A method for performing multiplexed,single-cell analysis in a microfluidic device, the method comprising:(i) loading a cell sample into a device, wherein the device comprises aplurality of holding chambers; (ii) holding a portion of the cell samplewithin each holding chamber, thereby providing a held cell; (iii)incubating the held cell with a first protein label in each holdingchamber, wherein the first protein label is configured to detect a firsttarget protein, thereby providing a labeled cell; (iv) incubating thelabeled cell with a first nucleic acid label, wherein the first nucleicacid label is configured to detect a first target nucleic acid, therebyproviding a multiplexed-labeled cell; (v) detaching themultiplexed-labeled cell, thereby providing a detached, intact cell; and(vi) performing an on-chip flow cytometry assay of the detached, intactcell, thereby performing multiplexed, single-cell analysis for thetarget protein and/or the target nucleic acid.
 2. The method of claim 1,further comprising: stimulating the cell sample with a stimulant.
 3. Themethod of claim 1, wherein the first protein label and/or the firstnucleic acid label further comprises a detectable marker.
 4. The methodof claim 1, wherein the first nucleic acid label further comprises afirst affinity agent, and the method further comprises, after step (iv),incubating the multiplexed-labeled cell with a secondary labelcomprising a second affinity agent, wherein the first and secondaffinity agents are configured to bind together.
 5. The method of claim4, wherein the method further comprises incubating themultiplexed-labeled cell with one or more tertiary labels, wherein eachtertiary label is, independently, configured to bind to the secondarylabel and independently comprises a proximity ligation probe; andincubating with one or more quaternary labels, wherein each quaternarylabel is, independently, configured to bind to a portion of theproximity ligation probe.
 6. The method of claim 1, further comprising:amplifying a signal of the first nucleic acid label.
 7. The method ofclaim 6, wherein the amplifying step comprises performing a rollingcircle amplification by providing one or more affinity agents configuredto bind to the first nucleic acid label, one or more proximity ligationprobes configured to bind at least one affinity agent, one or moreconnector probes configured to bind at least one proximity ligationprobe and to form a circular template, and one or more enzymesconfigured to generate a concatemer based on the circular template. 8.The method of claim 1, further comprising: incubating the labeled cellwith a second protein label in each assay chamber, wherein the secondprotein label is configured to detect a second target protein.
 9. Themethod of claim 8, wherein the first target protein is a cell surfaceprotein and the second target protein is an intracellular protein. 10.The method of claim 1, wherein step (vi) comprises hydrodynamicallyfocusing the detached, intact cell to a center of the device.
 11. Themethod of claim 1, further comprising: incubating the held cell with apost-translation modification label or a glycosylation label.
 12. Themethod of claim 1, wherein the plurality of holding chambers comprises aplurality of individually addressable holding chambers.
 13. The methodof claim 12, wherein each of the plurality of individually addressableholding chambers is connected to a programmable valve and a pump. 14.The method of claim 1, wherein the first nucleic acid label is a miRNAlabel or a mRNA label.
 15. The method of claim 1, wherein the firstprotein label is a cytosolic protein label, a cell surface receptorlabel, a cell surface protein label, a post-translational modificationlabel, a dynamic glycosylation label, an intracellular protein label, ora phospho-protein label.
 16. The method of claim 1, wherein step (iv)comprises conducting a fluorescent in situ hybridization assay.
 17. Themethod of claim 16, wherein the fluorescent in situ hybridization assaycomprises locked nucleic acid probes.